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WO2003038441A2 - Procedes de criblage a base de proteines de la famille des cited - Google Patents

Procedes de criblage a base de proteines de la famille des cited Download PDF

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Publication number
WO2003038441A2
WO2003038441A2 PCT/GB2002/004997 GB0204997W WO03038441A2 WO 2003038441 A2 WO2003038441 A2 WO 2003038441A2 GB 0204997 W GB0204997 W GB 0204997W WO 03038441 A2 WO03038441 A2 WO 03038441A2
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Prior art keywords
protein
cited
tfap2
amino acid
cbp
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PCT/GB2002/004997
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WO2003038441A3 (fr
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Jose Braganca
Fatima Marques
Shoumo Bhattacharya
Simon Bamforth
Helen Catherine Hurst
Jyrki Johannes Eloranta
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Isis Innovation Ltd
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Publication of WO2003038441A3 publication Critical patent/WO2003038441A3/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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • the present invention relates to the identification of novel interactions between the CITED family of transcription transactivator proteins and the TFAP2 family of transcription factors .
  • CBP/p300 Interacting Transactivators with ED-rich termini family of transcription transactivator proteins are characterized by a conserved 32 amino acid sequence at the C-terminus that is necessary and sufficient for binding p300/CBP (Bhattacharya, S. et al., Genes Dev. 13: 64-75, 1999) .
  • Members of the family include CITED1 (also known as Msgl) and CITED2 (also known as Mrgl or p35srj ) .
  • CITED family proteins co-activate TFAP2 identifies the CITED/TFAP2 interaction as a target for therapeutic intervention in the treatment of cancer, particularly breast cancer.
  • CITED proteins act synergistically with p300/CBP and each are independently required for full transcriptional activation via a TFAP2 protein thus providing an additional target for therapeutic intervention in treatment of cancers, particularly breast cancer and other conditions mediated by inappropriate transcriptional activation via the TFAP2 protein.
  • an in vitro method for identifying compounds which block or enhance the interaction between a CITED family protein and a TFAP2 protein and therefore have potential pharmacological activity in the treatment of diseases or conditions mediated by inappropriate interactions between said CITED and TFAP2 protein comprises: forming a mixture comprising a first protein component comprising a CITED protein, a second protein component comprising a TFAP2 protein, and a candidate compound, incubating the mixture under conditions which, in the absence of the candidate compound,, would permit binding of the CITED protein to the TFAP2 protein, and detecting any binding of the CITED protein to the TFAP2 protein; wherein compounds which block or stimulate binding of the CITED protein to the TFAP2 protein are identified as having potential pharmacological activity in the treatment of diseases or conditions mediated by inappropriate interactions betwee said CITED and TFAP2 proteins.
  • the mixture may include instead of the TFAP2 protein, a p300/CBP protein as the second protein component and CITED protein, to identify compounds which modulate the interaction between the CITED and p300/CBP proteins.
  • the candidate compound is one which blocks the interaction between said CITED and TFPA2 proteins, or CITED and p300/CBP proteins and which compo ' und may be useful in the therapeutic treatment of cancer and particularly breast cancer.
  • the invention further provides a cell-based a method for identifying compounds which modulate the interaction between a CITED prote ' in and a TFAP2 protein, which method comprises: providing a host cell containing a DNA construct comprising a reporter gene or a counter-selectable marker gene operably linked to a promoter regulated by a transcription factor having a DNA binding domain and an activating domain; expressing in said host cell a first hybrid DNA sequence encoding a first hybrid protein comprising a CITED protein fused in-frame to either the DNA binding domain or the activating domain of the said transcription factor; expressing in said host cell a second hybrid DNA sequence encoding a second hybrid protein comprising a TFAP2 protein fused in-frame to either the DNA binding domain or the activating domain of the said transcription factor, such that when the first fusion protein comprises the activation domain of the said transcription factor the second fusion protein comprises the DNA binding domain of the said transcription factor and when the first fusion protein comprises the DNA binding domain of the transcription factor the second fusion protein
  • the CITED protein is human CITED4 or human CITED2 and the TFAP2 protein is human TFAP2A, TFAP2B or TFAP2C.
  • the invention further provides a method of identifying compounds which are capable of down- regulating expression of a CITED protein from its natural promoter and therefore have potential pharmacological activity in the treatment of cancer, which method comprises:
  • the CITED protein may be replaced by any one of p300/CBP protein which also have been shown to play an integral part in transcriptional activation via TFPA2.
  • the cancer to be treated is breast cancer.
  • the CITED gene is the human C ' ITED4 gene or the human CITED2 gene ,
  • the invention further provides the following mutant CITED protein which lacks the ability to co- activate TFAP2 for use as therapeutic agents in the treatment of breast cancer:
  • mutant CITED4 polypeptide comprising amino acid residue numbers 21-184 of the amino acid sequence illustrated in Figure 4 but lacking amino acid residue numbers 1-20;
  • mutant CITED4 polypeptide comprising amino acid residue numbers 2-137 of the amino acid sequence illustrated in Figure 4 but lacking amino acid residue numbers 138-184;
  • mutant CITED4 polypeptide comprising amino acid residue numbers 21-137 of the amino acid sequence illustrated in Figure 4 but lacking amino acid residue numbers 1-20 and 138-184;
  • mutant CITED2 polypeptide comprising amino acid residue numbers 1-214 of the amino acid sequence illustrated in Figure 6 but lacking' amino acid residue numbers 215-270.
  • the invention further provides pharmaceutical compositions comprising at least one mutant CITED proteins as defined above plus a pharmaceutically acceptable carrier, diluent or excipient.
  • the invention further provides expression vectors encoding the mutant CITED proteins and for use of such vectors in somatic gene therapy.
  • TFAP2 is overexpressed in many breast cancer cell lines (Bosher, J. M. et al., Proc. Natl. Acad. Sci. USA, 92: 744-747, 1995). TFAP2 activates erbB2 and erbB3 gene transcription (Turner, B.C. et al., Cancer Res. 58: 5466-5472, 1998; Bosher, J.M. et al., Oncogene. 13: 1701-1707, 1996; Bosher, J.M. et al., Proc. Natl. Acad. Sci.
  • erbB2 is the target for a therapeutically very effective antibody (Herceptin) used in the treatment of breast cancer.
  • TFAP2 activates VEGF transcription (Berra, E. et al., Biochem Pharmacol. 60: 1171-8, 2000; Gille, J. et al., EMBO J. 16: 750-9, 1997).
  • blocking TFAP2 is predicted to inhibit angiogenesis.
  • TFAP2 activates TGF- ⁇ transcription (Wang, D. et al., J Biol Chem. 272: 14244-50, 1997).
  • TGF- ⁇ itself plays a major role in breast cancer (Humphreys, R.C. and Hennighausen, L. Oncogene.
  • the present invention provides screening methods for use in the identification of compounds which interfere with the interaction between CITED proteins, p300/CBP and TFAP2 proteins and therefore have potential pharmacological activity in the treatment of cancer and particularly breast cancer.
  • assay methodologies including labelled in vitro protein-protein binding assays and also cell-based assays, such as two- and three-hybrid screens.
  • a typical in vitro binding assay involves forming an assay mixture comprising a first protein component comprising a CITED protein, a second protein component comprising TFAP2 or p300/CBP and a candidate compound.
  • the protein components will typically comprise purified recombinant CITED, p300/CBP and TFAP2.
  • the protein components may further be labelled with a label which is directly or indirectly detectable, for example a radioactive label such as 35 S, a fluorescent or luminescent label, an enzyme or an epitope tag to facilitate the specific detection of one or other of the protein components.
  • Other components of the assay mixture might include, as appropriate, salts, buffer components etc to facilitate optimum protein-protein binding and to reduce background or non-specific interactions of the reaction components.
  • a plurality of assay mixtures are run in parallel, containing varying concentrations of the candidate compound.
  • One of these mixtures may contain zero candidate compound to serve as a negative control.
  • An appropriate positive control would typically also be included.
  • the assay mixture is incubated under conditions which, in the absence of the candidate compound, would allow binding of CITED to p300/CBP and/or TFAP2.
  • Assay parameters e.g. order of addition of the reaction components, time and temperature of the incubation, buffer composition of the assay mixture etc, may be readily optimised by routine experimentation, as would be well known to one " of ordinary skill in the art.
  • the incubation time should ideally be kept to a minimum.
  • CITED p300/CBP CITED p300/CBP to TFAP2
  • any convenient method If one or other of the protein components carries a label which is directly or indirectly detectable then specific binding of CITED p300/CBP to TFAP2 can be detected by detecting the presence of the label. Detection of one or other of the protein components may also be accomplished using a specific antibody, e.g. anti-CITED, in an ELISA type assay.
  • In vitro binding assays may also commonly include a washing or separation step after incubation and prior to detection of specific binding in order to separate bound from unbound reaction components, although for the purposes of high-throughput screening it is advantageous to keep any washing stages to a minimum.
  • a variety of means can be used to effect separation of bound from unbound components.
  • at least one of the protein components may be immobilised on a solid support, for example a microbead, a resin particle or the wells of a microtitre assay plate.
  • Separation may then be effected by, for example, washing the wells of the microtitre plate with a wash solution, washing microbeads or resin particles and then separating them from the bulk solution by centrifugation or, where the support is a magnetic bead, with the use of a magnet.
  • Protein components may be simply adsorbed onto the wells of a microtitre plate.
  • Protein components may also be linked to a solid support or matrix via a high affinity specific binding reaction, for example using the binding pairs biotin/avidin, biotin/streptavidin or GST/glutathione.
  • the protein component may be advantageously synthesised as a fusion protein containing one component of the binding pair, the other component of the binding pair being linked to the solid support.
  • Expression vectors suitable for use in the synthesis of biotinylated fusion proteins and GST tagged proteins are available commercially (e.g. PinPointTM system from Promega Corp.; pGEX system from Amersham Pharmacia Biotech) .
  • the CITED protein is radioactively labelled, for example by in vi tro translation in a cell-free system incorporating an 35 S-labelled amino acid.
  • the TFAP2 protein is synthesised as a recombinant GST-fusion (i.e., synthesised in E. coli using an expression vector, such as pGEX, adapted for the expression of an in- frame GST fusion protein) , purified and immobilised on glutathione agarose beads (SIGMA) .
  • the immobilised TFAP2 is then mixed with 3S S-labelled CITED in an aqueous solution in the presence of the candidate compound. Specific binding of CITED to TFAP2 is assayed by SDS-PAGE and autoradiography of bound CITED.
  • Assay 2 Assay 2
  • one of the protein components preferably the TFAP2 protein
  • a solid support containing a scintillant preferably the TFAP2 protein
  • the other protein component CITED
  • the two components are mixed in aqueous solution in the presence of the candidate compound. Specific binding of CITED to TFAP2 is determined by detecting the amount of light emission from the scintillant.
  • This method is based on the scintillation proximity assay (SPATM) from Amersham, which is widely used in high-throughput screening.
  • SPA beads linked to the first protein component are incubated for 30 minutes to one hour with a sample containing the radioactively labelled second protein component.
  • the radioactivity emitted by the labelled protein is brought into close proximity with the bead containing scintillant and therefore induces light emission from the scintillant.
  • the free labelled protein in the sample (non-bound) will not be held in sufficiently close proximity to the beads to induce light emission. Compounds which disrupt the binding of the interacting proteins will cause a decrease in the amount of light emitted during the experiment.
  • this assay may be carried out in either orientation, i.e. using CITED linked to the solid support containing scintillant and a radioactively labelled TFAP2 protein or using TFAP2 protein linked to the solid support containing scintillant and a radioactively labelled CITED.
  • Assay 3 In this assay the first protein component (usually that comprising the CITED protein) is linked to a label which is directly or indirectly detectable.
  • the second protein component (the TFAP2 protein) is immobilised on a solid support or matrix, such as a microbead or the well of a microtitre plate.
  • the two protein components are mixed in an aqueous solution in the presence of a candidate compound and specific binding of CITED to TFAP2 is determined by detecting the presence or absence of the label.
  • epitope tags such as HA, MYC, FLAG, GST and His tags, which are detectable using a specific antibody is particularly preferred.
  • the assay may use a directly detectable fluorescent label, such as GFP or any of the other autonomously fluorescent proteins known in the art.
  • a further assay may be based on the use of fluorescence energy transfer (FRET) , a technique well known in the art for the detection and quantitative measurement of a whole range of specific binding interactions in biological systems, to screen for compounds which modulate the binding of CITED to TFAP2.
  • FRET fluorescence energy transfer
  • FRET fluorophore
  • the fluorescence emission spectrum of the donor fluorophore overlaps with the absorption spectrum of the acceptor fluorophore, such that when the two components of the binding pair bind to each other, bringing the donor and acceptor fluorophores into close proximity, a proportion of the fluorescent signal emitted by the donor fluorophore (following irradiation with incident radiation of a wavelength absorbed by the donor fluorophore) will be absorbed by the proximal acceptor fluorophore (a process known in the art as fluorescence energy transfer) with the result that a proportion of the fluorescent signal emitted by the donor fluorophore is quenched and, in some instances, that the acceptor fluorophore emits fluorescence.
  • Fluorescence energy transfer will only occur when the donor and acceptor fluorophores are brought into close proximity by the specific binding reaction. Thus, in the presence of a compound which disrupts the specific binding, the amount of quenching is reduced resulting in an increase in the intensity of the fluorescent signal emitted by the donor fluorophore or a fall in the intensity of the signal emitted by the acceptor fluorophore) .
  • Suitable pairs, of donor and acceptor fluorophores are well known in the art.
  • a preferred combination comprises fluorescein as donor fluorophore and rhodamine as acceptor. Techniques for the conjugation of these fluorophores to proteins and peptides are well known in the art.
  • FRET-based assays are usually performed in vitro but a similar approach may be used in vivo in a suitable host cell, for example a mammalian cell line.
  • An in vivo assay may be based on the use of genetically encoded donor and acceptor fluorophores which can be expressed as fusion proteins fused in frame to CITED/p300/CBP and to TFAP2. This can be readily accomplished by transforming or transfecting the cell or organism with appropriate expression vectors arranged to express the fusion proteins.
  • the genetically encoded donor and acceptor proteins may be autonomous fluorescent proteins, for example GFP variants or GFP homologues, which exhibit different fluorescent properties and which have suitably overlapping emission/absorption spectra.
  • CITED protein encompasses members of the CITED (CBP/p300 Interacting Transactivators with ED-rich termini) family of transcription transactivator proteins are characterized by a conserved 32 amino acid sequence at the C-terminus that is necessary and sufficient for binding p300/CBP (Bhattacharya, S. et al., Genes Dev. 13: 64-75, 1999). Particularly preferred are CITED2 (Bhattacharya, S. et al., Genes Dev. 13: 64-75, 1999) and CITED4 (also known as HCITEDX and described in WO 01/68846) .
  • TFAP2 encompasses all TFAP2 isbfor s which interact with CITED proteins, including the human TFAP2 isoforms TFAP2A (Williams, T. et al .
  • the assays of the invention may also be carried out using natural occurring allelic variants (several of which are identified in publicly accessible databases such as GenBank and dbSNP) or synthetic variants, mutant forms or sub-fragments of TFAP2 which retain the ability to interact with CITED proteins. References in this context to "TFAP2" are to be construed accordingly.
  • candidate compounds may be tested using the method of the invention to determine whether they are capable of modulating the interaction between any of CITED, p300/CBP and TFAP2.
  • the compound may be of any chemical formula and may be one of known biological or pharmacological activity, a known ⁇ ompound without such activity or a novel molecule such as might be present in a combinatorial library of compounds.
  • Compounds which prevent or disrupt the interaction between any of CITED, p300/CBP and TFAP2 are identified as having potential pharmacological activity in the treatment of cancer and particularly breast cancer. Such compounds may be used as lead compounds in the further development of drugs with pharmaceutical potential or may themselves be formulated into pharmaceutical compositions.
  • Cell-based screening methods may be based upon "classical" two-hybrid methodology (first described in yeast cells by Chien et al . , Proc. Natl. Acad. Sci. USA., 88, 9578-9582, 1991).
  • a typical screen might be based on the use of a lacZ reporter gene under the control of the gal4 promoter, the read-out of the screen being ⁇ -galactosidase activity which can be easily measured using an appropriate fluorescent or luminescent substrate.
  • Cell-based screening assays can readily be adapted to be performed in a mid-to-high throughput screening format in multi-well microtitre assay plates. Host cells transfected. with the two fusion constructs are incubated with a candidate compound and the effect of the compound on the read-out of the assay (e.g. reporter gene expression) is recorded. Compounds which decrease the read-out of the assay would be scored as reducing the CITED/p300/CBP/TFAP2 interaction, for example either by preventing binding or by disrupting existing binding.
  • the first reverse two-hybrid system utilizes a yeast strain, which is resistant to cycloheximide due to the presence of a mutant CYH2 gene.
  • This strain also contains the wild-type CYH2 allele under the transcriptional ' control of the GAL1 promoter. Expression of the wild-type GAL4 protein is sufficient to restore growth sensitivity to cycloheximide. Growth sensitivity towards cycloheximide is also restored by the co-expression of the avian c-Rel protein and its I ⁇ B- ⁇ counterpart, p40, as GAL4 fusion proteins.
  • URA3 which encodes orotidine-5 ' -phosphate decarboxylase, an enzyme required for the biosynthesis of uracil.
  • URA3 + phenotype media lacking uracil
  • the ura3-encoded decarboxylase can also catalyze the conversion of a non-toxic analogue, 5-fluorooritic ⁇ acid (FOA) into a toxic product, 5-fluorouracil (Boeke et al., 1984, Mol. Gen. Genet. 197:345-346).
  • mutations that prevent an interaction can be selected from large libraries or random alleles.
  • molecules that dissociate or prevent an interaction could be selected from large libraries of peptides or compounds (Vidal et al., 1996, PNAS 93: 10315-10 " 320; Vidal et al., 1996, PNAS 93:10321-10326).
  • a third reversed yeast two-hybrid is based on the GAL80 gene as relay gene.
  • GAL80 encodes a protein that binds to and masks the activation domain of a transcriptional activator, such as GAL4.
  • the reporter genes which will provide the transcriptional read-out (HIS3 or LACZ), are dependent upon the functional GAL4 for expression. Only when the level of GAL80 masking protein is reduced by interfering with the two-hybrid interaction, will Gal4 function as a transcriptional activator, providing a positive transcriptional read-out for molecules that inhibit the two-hybrid protein-protein interaction.
  • the invention further provides methods of screening for compounds which down-regulate CITED or p300/CBP protein expression and therefore have potential therapeutic activity in the treatment of breast cancer.
  • a DNA construct in which the promoter region of a CITED or p300/CBP gene is positioned to control expression of a reporter gene encoding a protein product which is. directly or indirectly detectable.
  • the juxtaposition of the relevant promoter region and a reporter gene may be referred to herein as a "reporter gene expression construct”.
  • Reporter genes which may be used in accordance with the invention include those which encode a fluorescent product, such as green fluorescent protein (GFP) or other autonomous fluorescent proteins of this type or those which encode an enzyme product, such as for example chloramphenicol acetyl transferase (CAT), ⁇ -galactosidase and alkaline phosphatase, which is capable of acting on a substrate to produce a detectable product.
  • a fluorescent product such as green fluorescent protein (GFP) or other autonomous fluorescent proteins of this type or those which encode an enzyme product, such as for example chloramphenicol acetyl transferase (CAT), ⁇ -galactosidase and alkaline phosphatase, which is capable of acting on a substrate to produce a detectable product.
  • GFP green fluorescent protein
  • CAT chloramphenicol acetyl transferase
  • ⁇ -galactosidase alkaline phosphatase
  • Reporter gene assays using reporter gene expression constructs are well known in the art and commonly used in the art to test the promoter activity of a given DNA fragment. They may also be adapted, as in the present invention, to screen for compounds capable of modulating gene expression.
  • the reporter gene expression construct is preferably incorporated into a replicable expression vector so that it may be conveniently introduced into the eukaryotic host cell.
  • the eukaryotic host cell must be one which contains the appropriate transcription machinery for RNA Polymerase II transcription, and is preferably a ⁇ cultured mammalian cell.
  • the host cell is a cell type which is known to express the CITED or p300/CBP proteins in vivo or is a transformed cell line derived from a cell type known to express the CITED or p300/CBP protein.
  • An expression vector may be inserted into the host cell in a manner which allows for transient transfection or alternatively may be stably integrated into the genome of the cell (i.e. chromosomal integration) .
  • Chromosomal integration is generally preferred for drug screening because the expression constructs will be maintained in the cell and not lost during cell division, also there is no need to separately control for the effects of copy number.
  • Stable integration of a reporter gene expression construct into the genome of eukaryotic host cell may be achieved using a variety of known techniques. The most simple approach is selection for stable integration following transfection of a host cell with a plasmid vector. Briefly, a plasmid vector comprising a reporter gene expression construct consisting of the CITED or p300/CBP promoter region ligated to a promoterless reporter gene cDNA and also a gene encoding a dominant selectable marker, such as neomycin phosphotransferase, is first constructed using standard molecular biology techniques. The plasmid vector is then used to transfect eukaryotic host cells using one of the standard techniques such as, for example, lipofection.
  • a plasmid vector comprising a reporter gene expression construct consisting of the CITED or p300/CBP promoter region ligated to a promoterless reporter gene cDNA and also a gene encoding a dominant selectable marker, such as neomycin
  • Plasmid vectors suitable for use in the construction of stable cell lines are commercially available (for example the pCI-neo vector from Promega corporation, Madison WI, USA) .
  • Stable integration into mammalian chromosomes may also be achieved by homologous recombination, a technique which has been commonly used to achieve stable integration of foreign DNA into embryonic stem cells as a first stage in the construction of transgenic mammals.
  • Stable integration into eukaryotic chromosomes can also be achieved by infection of a host cell with a retroviral vector containing the appropriate reporter gene expression construct.
  • Reporter gene assays may be performed using promoter regions of the human CITED4 or CITED2 genes.
  • promoter region of the human CITED4 gene may refer to the complete sequence shown in Figure 8 or a fragment thereof lacking sequences downstream of the transcription initiation site or a transcriptionally active fragment of the sequence shown in Figure 8, for example a fragment of the complete sequence shown in Figure 8 which retain the ability to direct a tissue-specific pattern of gene expression, most preferably a tissue-specific gene expression pattern substantially identical to the native CITED4 gene expression pattern.
  • promoter region of the human CITED4 gene also encompasses the CITED4 proximal promoter region, this being defined as sequences immediately upstream of the CITED transcription start site which are necessary for correctly positioning RNA polymerase and directing an appropriate frequency of transcription initiation from the natural transcription start site, and also an artificial construct comprising the proximal promoter region plus any additional sequence elements which may be involved in regulating CITED gene expression, e.g. upstream enhancer elements etc.
  • the promoter and/or enhancer activity of fragments of the sequence shown in Figure ' 8 can be easily tested using reporter gene assays, for example by constructing a deletion series of the complete fragment.
  • Plasmid vectors containing reporter genes for use in testing the promoter and/or enhancer activity of DNA fragments are available commercially (e.g. the pGL2 and pGL3 vector series from Promega Madison, WI, USA) .
  • Promoter elements required for positioning of RNA polymerase and initiation of basal transcription are likely to be found immediately upstream of the transcription initiation site, whereas enhancer elements and elements required for tissue- specific expression might be found far upstream.
  • promoter region of the human CITED2 gene may refer to the complete sequence shown in Figure 9 or a fragment thereof lacking sequences downstream of the transcription initiation site or a transcriptionally active fragment of the sequence shown in Figure 9.
  • a fragment containing just promoters A and B i.e. deleted for sequences upstream of the BamHI site at -1037 retains promoter activity, albeit at a lower level than a fragment containing promoters A, B and C (Leung et al . , Genomics, 61, 307- 313, 1999) .
  • the compound may be of any chemical formula and may be one of known biological or pharmacological activity, a known compound without such activity or a novel molecule such as might be present in a combinatorial library of compounds.
  • the method of the invention may be easily adapted for screening in a medium-to-high throughput format .
  • Compounds which are identified as being capable of up-regulating CITED or p300/CBP gene expression should be further tested in order to establish whether the effect on gene expression is CITED-specific or non-specific. This could be achieved using a control cell containing a control reporter gene expression construct with no CITED promoter sequences.
  • the identification of the CITED/p300/CBP/TFAP2 interaction as a potential therapeutic target in the treatment of breast cancer leads to the development of therapeutic agents which specifically block, inhibit or disrupt the CITED/p300/CBP/TFAP2 interaction.
  • therapeutic agents may be based on mutant or variant CITED or p300/CBP proteins which do not co-activate TFAP2.
  • mutant or variant CITED p300/CBP proteins may be used as "dominant negative" agents to interfere with the interaction between the endogenous CITED or p300/CBP proteins and TFAP2 in vivo .
  • mutant CITED proteins which lack the ability to co-activate TFAP2 have been developed by the inventors.
  • a mutant CITED2 protein deleted for amino acid residues 215-270 is also unable to co-activate TFAP2. Therefore, the invention provides the following mutant CITED proteins for use as active therapeutic agents for the treatment o.f breast cancer: a mutant CITED4 polypeptide comprising amino acid residue numbers 21-184 of the amino acid sequence illustrated in Figure 4 but lacking amino acid residue numbers 1-20;
  • mutant CITED4 polypeptide comprising amino acid residue numbers 2-137 of the amino acid sequence illustrated in Figure 4 but lacking amino acid residue numbers 138-184; '
  • mutant CITED4 polypeptide comprising amino acid residue numbers 21-137 of the amino acid sequence illustrated in Figure 4 but lacking amino acid residue numbers 1-20 and 138-184;
  • mutant CITED2 polypeptide comprising amino acid residue numbers 1-214 of the amino acid sequence illustrated in Figure 6 but lacking amino acid residue numbers 215-270.
  • the invention therefore provides pharmaceutical compositions comprising a mutant CITED protein as defined above, together with a pharmaceutically acceptable diluent, carrier or excipient.
  • the mutant CITED proteins may be obtained by expression of the corresponding nucleotide sequence in a recombinant expression system or may be synthesised in a cell-free in vitro translation system (e.g. a reticulocyte lysate) .
  • a cell-free in vitro translation system e.g. a reticulocyte lysate
  • Suitable nucleotide sequences which encode the mutant proteins are as follows :-
  • Mutant CITED4 polypeptide comprising amino acid residue numbers 21-184 of the amino acid sequence illustrated in Figure 4 but lacking amino acid residue numbers 1-20 is encoded by a polynucleotide having the sequence shown from position 61 to 552 of Figure 5 but lacking the sequence from position 1 to 60.
  • Mutant CITED4 polypeptide comprising amino acid residue numbers 2-137 of the amino acid sequence illustrated in Figure 4 but lacking amino acid residue numbers 138-184 is encoded by a polynucleotide having the sequence shown from position 4 to 411 of Figure 5 but lacking the sequence from position 412 to 552.
  • Mutant CITED4 polypeptide comprising amino acid residue numbers 21-137 of the amino acid sequence illustrated in Figure 4 but lacking amino acid residue numbers 1-20 and 138-184 is encoded by a polynucleotide having the sequence shown from position 61 to 411 of Figure 5 but lacking the sequences from position 1 to 60 and from position 412 to 552.
  • Mutant CITED2 polypeptide comprising amino acid residue numbers 1-214 of the amino acid sequence illustrated in Figure 6 but lacking amino acid residue numbers 215-270 is encoded by a polynucleotide having the sequence shown from position 1 to 644 of Figure 5 but lacking the sequence from position 645 to 812.
  • compositions comprising mutant CITED proteins may be formulated for administered by any conventional route, including injection or infusion.
  • the mutant CITED protein may be administered in commonly used intravenous fluid (s) , for example physiological saline, Ringer's solution or 5% dextrose, and administered by direct injection or infusion.
  • s intravenous fluid
  • Other routes of administration of a mutant CITED protein- based drug are also contemplated, e.g. transdermal administration, inhalation or even oral delivery.
  • the therapeutic protein is linked, preferably by in-frame N-terminal fusion, to a protein transduction domain (PTD) which mediates transduction into mammalian cells in a process which is independent of specific receptors or transporters.
  • PTD protein transduction domain
  • the preferred PTD is an 11 amino acid region of the human immunodeficiency virus TAT protein having the amino acid sequence YGRKKRRQRRR (M. Green and P.M. Loewenstein. Cell, 55, 1179, 1988; A.D. Frankel and CO. Pabo. Ibid. , p 1189), although synthetic variants of this sequence having equivalent function, i.e. which mediate similar or enhanced levels of protein transduction compared to the wild-type sequence, may also be used.
  • compositions comprising a mutant CITED protein linked to a protein transduction domain, preferably by in-frame N-terminal fusion, and also medicaments comprising these compositions together with appropriate pharmaceutically acceptable carriers, diluents or excipients.
  • Compositions containing the protein transduction domain-CITED composition suspended in physiological saline or saline plus 10% glycerol are suitable for in vivo use (Schwarze et al , ibid) .
  • the invention further provides for delivery of a therapeutically effective amount of a nucleic acid encoding a mutant CITED protein to cells either in vivo or ex vivo, in other words the invention provides for "gene therapy" using nucleic acid encoding the mutant CITED protein.
  • Nucleic acid encoding the mutant protein is placed into a suitable expression vector under operable control of regulatory elements which permit expression of the sequence in cells of the subject to be treated. Numerous transfection and transduction techniques as well as appropriate expression vectors for carrying out such procedures are well known in the art.
  • adenovirus-derived vectors or retroviral vectors are viral vectors, particularly adenovirus-derived vectors or retroviral vectors, although non-viral vectors,, for example plasmid expression vectors, have also been proposed for use in somatic gene therapy.
  • adenoviral vectors are known in the art.
  • WO 95/00655 describes Ad5 vector systems which are deleted in both the El and E3 regions.
  • Another useful adenovirus vector is pCMVAdTrack which is available commercially.
  • the present invention is further directed to inhibiting expression of endogenous CITED or p300/CBP proteins in vivo by the use of antisense technology, as a means of providing therapeutic treatment for breast cancer.
  • Antisense technology can be used to control gene expression through either triple-helix formation using an antisense DNA (or modified versions thereof) or inhibition of expression using an antisense RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA.
  • an antisense RNA oligonucleotide preferably from 10 to 40 base pairs in length, may be designed to be complementary to a portion of the coding region of CITED or p300/CBP, most preferably a region corresponding to the N-terminal part of the protein.
  • the antisense RNA oligonucleotide hybridises to the mRNA in vivo and blocks translation of an mRNA molecule into the Akt-3 (antisense - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1998) ) .
  • a DNA oligonucleotide may be designed to be complementary to a region of the gene involved in the initiation or regulation of transcription (triple-helix - see Lee et al . Nucl. Acids Res., 3:173 (1979); Cooney efc al . , Science, 241:456 (1988); and Dervan et al . , Science, 251: 1360 (1991) , thereby preventing transcription of CITED mRNA.
  • a pharmaceutical composition in accordance with this aspect of the invention may include a therapeutically effective amount of the antisense nucleic acid in combination with any standard physiologically and/or pharmaceutically acceptable carriers known in the art .
  • “Pharmaceutically acceptable” means a non-toxic material which does not interfere with the activity of the pharmaceutically active ingredients in the compositipn .
  • “Physiologically acceptable” refers to a non-toxic material that is compatible with a biological system such as a cell , tissue or organism.
  • Physiologically and pharmaceutically acceptable carriers may include diluents , fillers , salts, buffers , stabilizers , solubilizers etc .
  • the antisense nucleic acids described above can be delivered to cells by procedures known in the art such that the anti-sense RNA or DNA may be expressed in vivo to inhibit production of CITED or p300/CBP in the manner described above.
  • Antisense compositions according to the invention would be used to reduce or inhibit the expression of CITED p300/CBP protein in vivo and therefore block the interaction in TFAP2.
  • the pharmaceutical preparations of the invention are to be administered in pharmaceutically acceptable amounts, an effective amount being an amount of a pharmaceutical preparation that alone, or together with further doses, produces the desired response in the condition being treated.
  • an effective amount being an amount of a pharmaceutical preparation that alone, or together with further doses, produces the desired response in the condition being treated.
  • the precise amount of the composition administered will, however, generally be determined by a medical practitioner, based on the circumstances pertaining to the disorder to be treated, such as the severity of the symptoms, the composition to be administered, the age, weight, and response of the individual patient and the chosen route of administration.
  • FIG. 1 illustrates that CITED2 physically and functionally interacts with TFAP2, a, In vitro binding assays.
  • Top The binding of 35 S-labelled TFAP2A, C and B isoforms respectively (generated by in vitro translation) to bacterially produced GST (lanes 2, 7, 10), GST-CITED2 (lane 3, 8, 11), or GST-CITED2 ⁇ (expressing residues 1-215, lane 4, 9, 12), immobilized on glutathione-sepharose beads was tested. Lanes 1, 5 and 6 contain 10% of the input TFAP2 in vitro translate.
  • Bottom Coomassie strain showing relative amounts of GST proteins, b, Yeast two-hybrid assays showing the transcriptional activation of an integrated GAL4-2acZ reporter.
  • PGBT9-TFAP2 isofor s or control vector, and pGAD424-CT ⁇ D2 or pGAD424- CITED2A were co-transfected as indicated.
  • Results (mean of duplicate assays) are presented as relative lacZ activity, corrected for optical density of the yeast culture, c, Transcriptional activation of p3xAP2-Bluc reporter in Hep3B cells by co-transfected CMV- TFAP2A, CMV-TFAP2C, RSV- TFAP2B and control vectors, and effect of co-transfected CMV-CITED2 or vector control.
  • Results are presented as RLU (relative luciferase units, corrected for transfection efficiency) and show the mean +SEM of three independent experiments.
  • the control transfection value (at extreme left) is set at 1. d, Co-activation of p3xAP2-Bluc by co-transfected CMV- TFAP2A (60 ng) and increasing doses (0, 40, 80, 120 and 160 ng) of CMV-CITED2 and CVN-CITED2 ⁇ (a mutant lacking C- terminal residues 215 to 270).
  • the total dose of CMV plasmid in each transfection was kept constant using CMV-vector (pcDNA3) . Results are presented as RLU.
  • the control transfection value (at extreme left) is set at 1. e.
  • FIG. 2 Illustrates interactions of CITED proteins with TFAP2 isoforms. Top: The binding of 35 S-labelled TFAP2A (lanes 2-5), TFAP2B (lanes 7-10) and TFAP2C (lanes 12-15), to GST (lanes 2, 7, 12) or GST-CITEDl (lanes 3, 8, 13), or GST-CITED2 (lanes 4, 9, 14, or
  • FIG. 3 Illustrates that CITED4 co-activates TFAP2 isoforms.
  • A Transcriptional activation of p3xAP2-Blue reporter (40ng) in Hep 3B cells by co-transfected CMV- TFAP2a, CMV-TFAP2C, RSV-TFAP2B and control vectors (40 ng each), and the effect of co-transfected CMV-CITED4, CMV-CITED4 ⁇ (a mutant lacking the p300/CBP binding domain) or vector control (40ng each) .
  • Results are presented as RLU (relative luciferase units, corrected for transfection efficiency) and show the mean+SEM of three independent experiments.
  • the control transfection value (at extreme left) is set at 1.
  • Results are presented as in A.
  • C Transcriptional activation of p3xAP2-Bluc reporter (40ng) in Hep G2 cells by co- transected CAV.-TFAP2A, CAV.-TFAP2C, RSV-TFAP2B and control vectors (40 NG each) , and the effect of co- transected CAV.-CITED4, CAV.-CITED2, or vector control (40 ng each) .
  • Results are presented as in A.
  • Figure 4 Shows the amino acid sequence of CITED4 (HCITEDX) .
  • Figure 5 Shows the nucleotide sequence of CITED4 (HCITEDX) .
  • Figure 6 Shows the amino acid sequence of CITED2 (GenBank accession AF129290) . .
  • Figure 7 Shows the nucleotide sequence of CITED2 (GenBank accession AF 129290) .
  • Figure 8 Shows the nucleotide sequence of the 5' flanking region of the human CITED4 gene.
  • Figure 9 Shows the nucleotide sequence of the 5' flanking region of the human CITED2 gene. The positions of three putative promoters identified by the computer program NNPP are indicated (see Leung et al . , Genomics, 61, 307-313, 1999 for details). The sequence is numbered relative to the predicted transcription start site of promoter A (arrow) at +1.
  • Figures 10-12 Show amino acid sequences for the human TFAP2 isoforms TFAP2A (GenBank NM_003220), TFAP2B and TFAP2C and also nucleotide sequences encoding the proteins. Start and stop codons are underlined in the nucleotide sequences.
  • Figure 13 illustrates that p300 and CBP synergize with CITED2 to co-activate TFAP2A.
  • Results ( ean ⁇ SEM, three independent experiments) are presented as relative luciferase units (RLU) , corrected for lacZ activity. The control transfection value (at extreme left) is set at 1.
  • B Transcriptional co-activation of p3xAP2-Bluc reporter in Hep G2 cells using the conditions described in A. Results are presented as in
  • Figure 14 illustrates that TFAP2A transcriptional activity is impaired in mouse embryonic fibroblasts
  • MEFs lacking one allele of either p300 or CBP.
  • A Transcriptional activation of p3xAP2-Bluc reporter (80 ng) by co-transfected CMV-TFAP2 (isoforms A, B and C) or the control CMV-vector (40 ng each), and CMV-p300 or the control CMV-vector (200 ng each) in p300 +/+ and p300 +/- MEFs.
  • Results (mean+SEM, three independent experiments) are presented as relative firefly luciferase units (RLU) , corrected for renilla luciferase activity.
  • the control transfection value (at extreme left) is set at 1.
  • B Transcriptional activation of p3xAP2-Bluc reporter (80 ng) by co-transfected CMV-TFAP2 plasmids or the control CMV-vector (40 ng each) and CMV-CBP or the control CMV-vector (200 ng each) in CBP +/+ and CBP +/- MEFs. Results are presented as in A.
  • FIG. 15 illustrates that TFAP2A physically interacts with p300 in vivo. Extracts from U2-OS cells cotransfected with the indicated plasmids, were immunoprecipitated with the indicated monoclonal antibodies. 9E10 is an anti-myc antibody, RK5C1 an anti-GAL4 antibody and PAB419 is a control antibody. Lanes 1 to 6 contain 10% of the cell extracts used for immunoprecipitations shown in lanes 7-12. Panels A and C show anti-GAL4 immunoblots of the immunoprecipitation reactions. The position of GAL4- TFAP2A and GAL4 peptides are indicated. The asterisks in panel A indicate non-specific immunoprecipitating antibody related bands.
  • Panel B shows the anti-CITED2 immunoblot, and the position of CITED2 is indicated. A longer exposure of the input extracts is also presented below the main panel (lanes 1 to 6) to show endogenous CITED2.
  • Figure 16 illustrates that CITED2 is required -for the interaction between TFAP2Aand p300.
  • Hep3B cells were co- transfected with CMV-p300-E2 or CMV-p300DCHl-E2 (40 ng each), CMV-CITED2 or control plasmid (40 ng each), and CMV-VP16-TFAP2A or control plasmids (40 ng each), 3xE2- luc reporter (100 ng) and CMV-lacZ (100 ng) .
  • Results (mean+SEM, three independent experiments) are presented as relative luciferase units (RLU) , corrected for lacZ activity.
  • Cited2-/- transgenic mice in which the urine CITED2 homolog Cited2 is disrupted by homologous recombination. Deletion of Cited2 in these mice results in embryonic lethality. Cited2-/- embryos dies with cardiac malformations, adrenal agenesis and exencephaly or anterior open neural tube defects. Since CITED proteins function as transcription co-activators, the inventors hypothesised that the above-mentioned abnormalities may arise as a result of defective co- activation of a transcription factor required for neural crest and neural tube development.
  • Candidate transcription factors included Pax3, RAR (retinoic acid receptor) isoforms and Tfap2 (AP-2) isoforms.
  • the cloning of a CITED2 cDNA in a protein-interaction cloning experiment using a TFAP2C probe strongly suggested a link to Tfap2.
  • the CITED2 fragment identified in the above experiment i.e. the C- terminal residues 234-270, overlaps with the EP300/CREBBP-binding domain previously mapped to residues 224-255 (Bhattacharya et al . , Genes Dev, 13, 64-75, 1999) .
  • CITED2 binds other TFAP2 isoforms in vi tro binding assays. All isoforms of TFAP2 (generated by in vitro translation) bound specifically to immobilized GST-CITED2, but not to GST alone, or to GST-CITED2 ⁇ (a mutant lacking residues 215- 270, Fig. la) . The C-terminus of CITED2 is thus necessary for the interaction with TFAP2 isoforms in vitro . These observations were confirmed using a yeast two-hybrid strategy (Fig. Lb) .
  • GAL4 activation domain-tagged CITED2 (pGAD42 >-CITED2) was co-expressed with GAL4-DNA-binding domain-TFASP2 isoforms (pGBT9- TFAPA, B and C) , or vector control, in yeast cells containing an integrated . lacZ reporter downstream of GAL4 binding sites.
  • pGAD424 -CITED2 significant transactivation of the lacz reporter was observed with pGAD424 -CITED2.
  • No activation above background was observed with pGAD424-CITED2 ⁇ .
  • Hep 3B cells a human hepatocellular carcinoma cell line that has low levels of endogenous TFAP2 and CITED2
  • a luciferase reporter cloned downstream of TFAP2 binding elements p3xAP2-Bluc, Fig. lc
  • Transfection of vectors expressing TFAP2 isoforms activated this reporter.
  • Co- transfection of CMV-CITED2 further activated the reporter by 2.8 to 5.2 fold, depending on the TFAP2 isoform. Similar results were obtained in another human hepatocellular carcinoma cell line (Hep G2, data not shown) .
  • Co-activation of TFAP2A was dependent on the dose of co-transfected CMV-CITED2 (Fig. 5d) .
  • CITED2 ⁇ (Jacking residues 215-270) did not co-activate TFAP2A, indicating that these residues, which are necessary for interaction with TFAP2 and EP300/CREBBP in vitro, are also required for the co-activation effect.
  • similar results were obtained with TFAP2B and TFAP2C (data not shown) .
  • the activity of the reporter gene in Cited2 ⁇ l ⁇ MEFs was reduced significantly (to 45% of that seen in Cited2 ++ MEFs) , indicating that endogenous Tfap2 function in these cells is defective. Transfection of.
  • the retinoic acid signalling pathway, and the transcription factor Pax3, are also required for the normal neural crest development and neural tube closure (Le Douarin, N.M. & Kalcheim, C. The Neural Crest, Cambridge University Press, Cambridge, 1999; Juriloff, D.M. & Harris, M.J. Hum. Mol. Genet. 9, 993-1000, 2000).
  • a retinoic acid receptor reporter gene (DR5-tk-luc) was transfected into Cited2 ++ and Cited2 ⁇ ' ⁇ MEFs.
  • pcDNA3- FAP2A and pcDNA3- TFAP2C were generated from pRSV- FAP2B (Bosher, J.M. et al . , pncogene, 13, 1701- 1707, 1996) .
  • pncogene 13, 1701- 1707, 1996) .
  • S. cerevisiae strain SFY526 was used following the instructions of the manufacturer (Clontech) , and data was normalized to the optical density of the yeast culture.
  • TFAP2 isoforms and CITED2 were cloned into pGBT9 and pGAD424, respectively.
  • TFAP2 isoforms Vectors expressing human TFAP2 isoforms were used to. synthesise 35 S-labelled proteins by coupled in vitro transcription-translation. These peptides were tested for interaction with GST-CITED1, GST-CITED2, or GST- CITED4 fusion proteins (Fig. 2) . As shown previously, TFAP2 isoforms specifically and strongly interacted with GST-CITED2. They also interacted although to a lesser extent, with GST-CITED4, and interacted very weakly with GST-CITED1.
  • CITED4 co-activates TFAP2 isoforms in mammalian cells
  • CITED4 might also function as a TFAP2 co-activator.
  • Hep 3B cells a human hepatocellular carcinoma cell line that has low levels of endogenous TFAP2
  • a luciferase reporter cloned downstream of TFAP2 binding elements (p3xAP2-Bluc, Fig.. ' 3A)
  • Transfection of vectors expressing TFAP2 isoforms activated this reporter.
  • Co-transfection of CMV-CITED4 resulted in further activation of the reporter.
  • CMV-CITED4 did not activate the reporter in the absence of TFAP2.
  • CMV- CITED4 ⁇ which lacks the transactivation and CBP/p300- binding domain, did not co-activate TFAP2 in spite of being expressed to a similar level (Fig. 3D, and data not shown) .
  • CITED4 appeared to be a significantly stronger co-activator of all three TTFAP2 isoforms, but especially of TFAP2A and TFAP2B (Fig. 3B) .
  • TFAP2 co-activation assays we have found that HA-tagged CITED peptides behave like the respective untagged peptides (data not shown) .
  • HA-CITED4 was produced at a higher level than HA-CITED2, we transfected HA-tagged- CITED2 and CITED4 plasmids into Hep 3B cells and examined protein expression by western blotting (Fig. 3D) . The expression of HA-CITED4 was indeed significantly greater than HA-CITED2.
  • CITED2 and CITED4 are a relatively weak co-activator when compared to CITED2 (Fig. 3C) , in spite of having a relatively higher expression level (Fig. 3E) . This is especially true for TFAP2c, for which CITED2 appears to be a particularly good co-activator.
  • CBP histone acetyl transferase
  • mice lacking p300 or CBP develop neural tube closure, cardiac and skeletal defects (3-5) .
  • P300 and CBP have overlapping functions, as indicated by the fact that mice lacking one allele of p300 and one allele of CBP (double heterozygotes) show early embryonic lethality, with defects in neural tube closure. However, they also have clearly distinct functions as well. For instance, retinoid receptor function requires p300 rather than CBP, whereas CREB function requires CBP rather than p300 (5,7) .
  • the PHD finger of p300, but not CBP is essential for histone acetyl transferase activity (8) .
  • the first cysteine-histidine rich (CHI) region of p300/CBP directly binds members of a recently identified gene family termed CITED (CBP/p300 Interacting Transactivators with ED-rich termini) (9-12) .
  • Members of this family include CITED1 (also known as Msgl) (13) , CITED2 (splice isoforms known as Mrgl or p35srj(9,13- 15)), and CITED4(16).
  • Msgl also known as Msgl
  • CITED2 splice isoforms known as Mrgl or p35srj(9,13- 15)
  • CITED4(16) A key feature of this family is a conserved 32 amino acid sequence motif at the carboxy- terminus that is necessary and sufficient for binding p300/CBP(9,16) .
  • CITED proteins do not appear to bind DNA directly, but function as transcriptional co-activators (11,16-18
  • CITED2 is a ubiquitously expressed growth factor and hypoxia inducible gene with oncogenic properties (9,15). Nearly all cellular CITED2 is physically complexed with p300/CBP, indicating, that it binds with high affinity (9) . Mice lacking CITED2 have abnormal cardiac, adrenal, neural tube, and cranial ganglia (18- 20) . CITED2 (and CITED4) physically interact with, and co-activate isoforms A, B and C of the DNAbinding transcription factor AP-2 • (TFAP2) (16,18). CITED2 is necessary for TFAP2 transactivation, and embryos lacking CITED2 have reduced expression of the TFAP2 target gene ErbB3 (21) in the neural crest (18) .
  • TFAP2A and TFAP2B mutations also result in neural tube, cardiac, and cranial ganglia defects (22-26) , it is likely that abnormal embryonic development in Ci ted2 / mice results, at least in part, from defective TFAP2 function (18) .
  • Both CITED2 and intact p300 /CBP function are independently required for full transcriptional activation by TFAP2A.
  • Our data suggests that CITED2 co-activates TFAP2A, at least in part, by recruiting p300/CBP.
  • Plasmids - CMV-TFAP2A, B, and C and CMV-CITED2 were described previously (18) .
  • P3xAP2-Bluc gift from Helen Hurst, CRUK, Hammersmith Hospital, London
  • CMV-p300, CMV-CBP, ' CMVp300-E2, CMV- p300DCHl-E2 and the E2-luc reporter were gifts from Richard Eckner (University of Zurich, Switzerland) (29) .
  • CMV-p300DCHl-E2 lacks residues 346-510 of p300.
  • CMV- VP16-TFAP2A was a gift from Stefan Gaubatz (University of Marburg, Germany) .
  • CMV-p300-myc and CMV-p300 (D1399Y) - myc (30) were gifts from Tso-Pang Yao (Duke University, NC) .
  • CMV-GAL4 and CMV-lacZ were gifts from Ronald Evans (Salk Institute, San Diego, CA) .
  • pRL-CMV renilla luciferase gene attached to CMV promoter was from Promega.
  • CMV-GAL4-TFAP2A was generated by PCR, and sequenced to confirm, the absence of artifacts.
  • Antibodies - RK5C1 (Santa Cruz) is a monoclonal antibody against GAL4-DNA binding domain. Anti-CITED2 poiyclonal antibody has been described previously (9) . PAB419 and 9E10 are monoclonal antibodies against SV40 Tag and the myc-epitope respectively, and were gifts from Jim DeCaprio (Dana-Farber Cancer Institute, Boston MA) . Anti-tubulin antibody was obtained from Sigma (T- 5293) . Western blots were performed as previously described (9) .•
  • Immunoprecipitations - U2-OS cells were plated at 2 x 106 cells per plOO plate and transfected the following day with 10 ug of the indicated plasmids using Fugene 6 (Roche) . Cells were lysed, and immunoprecipitations were performedas described (9) in buffer containing Tris-HCl (pH8) 50mM, NaCl 150mM, NP40 0.5%, EDTA 0.5 mM, protease and phosphatase inhibitors (complete protease inhibitor (Roche), PMSF lmM, sodium orthovanadate 0.5 mM, sodium fluoride 5 mM) , and DTT 1 mM.
  • CMV control vector was added to each transfection mix so that the total plasmid amount and CMV promoter per transfection were constant for each experiment.
  • Amounts of DNA used per transfection refer to the amounts added per well of a 24 well plate.
  • Hep 3B cells a human hepatocellular carcinoma cell line that has low levels of endogenous TFAP2 and CITED.2
  • a luciferase reporter cloned downstream of TFAP2 binding elements p3xAP2-Bluc
  • vectors expressing TFAP2A, CBP or p300, CITED2 Fig. 13A
  • CMV-CBP or CMV-p300 showed no effect on reporter plasmid activity in the absence of co-transfected CMV-TFAP2A or CMV-CITED2.
  • MEFs mouse embryonic fibroblasts
  • Anti-myc immunoprecipitates from cells transfected with CMV-p300-myc and CMVGAL4-TFAP2A contained GAL4-TFAP2A (Fig. 15A, lane 7) and endogenous CITED2 proteins (Fig 15B, lane 7) .
  • Control primary antibody immunoprecipitates from the same cells did not contain GAL4-TFAP2A or CITED2.
  • the p300 - TFAP2A interaction requires CITED2 and an intact p300-CHl domain
  • CITED2 is involved in the recruitment of p300 to TFAP2A
  • a mammalian two- hybrid assay in Hep3B cells to detect interactions between the ⁇ bait' p300-E2 (a fusion of p300 with the bovine papilloma virus E2- DNAbinding domain) , and the ⁇ prey' VP16-TFAP2A (Fig. 16) .
  • a reporter plasmid containing E2 binding sites upstream of the luciferase gene Transfection of CMV- VP16-TFAP2A alone led to a weak ' activation of the reporter, and may reflect the presence of cryptic TFAP2 binding sites in the reporter.
  • CMV-VP16-TFAP2A led to a marginal increase in reporter activity.
  • CMV-p300-E2 there was no marked increase in reporter activity with co-transfected CMV-CITED2 (Fig. 16) J This indicates that the p300 - CHI domain is essential for the CITED2-induced interaction between TFAP2A and p300.
  • mice In mice, the lack of a single allele of p300 results in a significant incidence of embryonic lethality and neural tube defects (5) . Mutation of a single CBP allele in mice results in growth retardation, skeletal, hematopoietic, cardiovascular and adipose tissue defects (4,34-36). In humans, CBP mutations result in Rubinstein-Taybi Syndrome (6) .
  • Our experiments show that both p300 and CBP heterozygote MEFs display defects in transactivation by TFAP2. The successful rescue of these defects by ectopically expressed p300 and CBP indicates that they are the specific factors limiting transactivation by TFAP2 in these MEFs.
  • TFAP2A, CITED2 and p300 can interact in vivo using two independent methods.
  • TFAP2A and CITED2 were recovered in anti-p300 immune complexes from transfected cells, indicating that the three proteins can be physically associated in vivo.
  • the p300-CHl domain (which contains the CITED2-binding site within p300 (9)) is required for the mammalian two-hybrid interaction between TFAP2A and p300. This implies that CITED2 binding to p300 is necessary for the TFAP2A - p300 interaction.
  • TFAP2A and p300/CBP can interact both physically and functionally, and that these interactions - require CITED2. They also show that p300 /CBP and CITED2 are independently required for full transcriptional activation by TFAP2A. Our results imply that CITED2 co- activates TFAP2A, at least in part, by recruiting p300/CBP. In previous experiments we showed that CITED2 binds directly to ⁇ 300/CBP, and to TFAP2 isoforms independently of its interaction with p300 and CBP (9,18) . A simple model is that CITED2 acts as a bridge, linking TFAP2A to p300/CBP.
  • interactions with CITED2 may lead to modifications in p300/CBP and/or TFAP2A, allowing them to interact.
  • these results support the idea that physical and functional interactions between p300/C.BP, CITED2 and TFAP2A are essential for normal neural tube and cardiac development, and that certain aspects of p300 and CBP haploinsufficiency (Rubinstein-Taybi syndrome) result from defective TFAP2 function.

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Abstract

La présente invention concerne un procédé permettant d'identifier in vitro des composés bloquant l'interaction entre une protéine de la famille des CITED et une protéine TFAP2, ces composés présentant ainsi une activité potentielle pour le traitement du cancer. A cet effet, on commence par réaliser un mélange entre un premier composant à base de protéines dont une protéine CITED, un second composant à base de protéines dont une protéine TFAP2, et un candidat composé. On met à incuber le mélange dans des conditions qui, en l'absence du candidat composé, permettraient la liaison de la protéine CITED à la protéine TFAP2. Il ne reste plus qu'à détecter les éventuelles liaisons de la protéine CITED à la protéine TFAP2. En l'occurrence, les composés qui bloquent la liaison de la protéine CITED à la protéine TFAP2 sont identifiés comme présentant une activité pharmacologique potentielle dans le traitement du cancer du sein.
PCT/GB2002/004997 2001-11-02 2002-11-04 Procedes de criblage a base de proteines de la famille des cited WO2003038441A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
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EP1793003A1 (fr) * 2005-11-30 2007-06-06 Deutsches Krebsforschungszentrum, Stiftung des öffentlichen Rechts CITED4 en tant que marqueur de pronostic pour les tumeurs oligodendrogliales
JP2007275052A (ja) * 2006-03-17 2007-10-25 Hironobu Yanagimori ＴＢＣＥ遺伝子またはＣＩＴＥＤ２遺伝子に特異的なｓｉＲＮＡおよびその用途
US20130011422A1 (en) * 2003-05-16 2013-01-10 Neil Berinstein Tumor Antigens for the Prevention and/or Treatment of Cancer
US20200247861A1 (en) * 2012-04-02 2020-08-06 Modernatx, Inc. Modified polynucleotides for the production of oncology-related proteins and peptides

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GB0006572D0 (en) * 2000-03-17 2000-05-10 Isis Innovation Novel transcription transactivator protein
WO2003000730A1 (fr) * 2001-06-21 2003-01-03 The General Hospital Corporation Composes qui modulent les actions oestrogeniques

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130011422A1 (en) * 2003-05-16 2013-01-10 Neil Berinstein Tumor Antigens for the Prevention and/or Treatment of Cancer
EP1793003A1 (fr) * 2005-11-30 2007-06-06 Deutsches Krebsforschungszentrum, Stiftung des öffentlichen Rechts CITED4 en tant que marqueur de pronostic pour les tumeurs oligodendrogliales
WO2007062827A1 (fr) * 2005-11-30 2007-06-07 Deutsches Krebsforschungszentrum, Stiftung Des Öffentlichen Rechts Cited4 en tant que marqueur de pronostic dans des tumeurs oligodendrogliales
JP2007275052A (ja) * 2006-03-17 2007-10-25 Hironobu Yanagimori ＴＢＣＥ遺伝子またはＣＩＴＥＤ２遺伝子に特異的なｓｉＲＮＡおよびその用途
US20200247861A1 (en) * 2012-04-02 2020-08-06 Modernatx, Inc. Modified polynucleotides for the production of oncology-related proteins and peptides

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