Classifications

• • 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
  • C07K14/4703—Inhibitors; Suppressors
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates to methods for the modulation of nuclear receptor mediated processes, compounds useful therefor and methods for the identification and use of such compounds.
• RXR 9-cis retinoic acid receptor
• TR thyroid hormone receptor
• RAR retinoic acid receptor
• VDR vitamin D receptor
• PPAR prostanoids
• Transcriptional repression is an intrinsic part of endocrine physiology and contributes to feedback regulation associated with the inhibition of the physiologic response. Indeed, the thyroid hormone receptor is converted to an oncogene by mutations which block hormone binding and create a constitutive transcriptional repressor (Damm et al . EMBO J. 6:375-382
• SMRT and N-CoR Transcriptional co-repressors
• chromatin remodeling has been suggested to be a component of transcriptional regulation (for review see Wolffe and Pruss. Curr. Biol . 6:234-237 (1996) : Felsenfeld Cell 86:13-19 (1996)). Indeed, it has been suggested that specific transcriptional activation may be involved in local changes in chromatin structure. In fact, it has recently been demonstrated that nuclear hormone receptors may utilize the CREB binding protein
• CBP CBP
• p300 Janknecht and Hunter Nature 383:22-23 (1996)
• Chakravarti, et al . Nature 383:99-103 (1996); Hanstein et al . PNAS 93:11540-11545 (1996); Kamei et al . Cell 85:403-414 (1996); Yao et al . PNAS 93:10626-10631
• CBP/p300 harbors intrinsic histone acetyltransferase activity, resulting in alternative or perhaps simultaneous histone acetylation (Ogryzko et al .
• APL acute promyelocytic leukemia
• histone deacetylase associates with hormone receptor complexes and contributes to the repression thereof.
• histone deacetylase inhibitors relieves this repression.
• histone deacetylase inhibitors have been found to be useful for the activation of genes responsive to hormone receptors.
• formulations useful for modulation of hormone- mediated processes have been developed.
• assays have been developed for the identification of compounds useful to modulate the above-described processes.
• methods of employing such compounds to modulate or enhance hormone mediated processes such as human cancers.
• SMRT and components of the histone deacetylase complex have been discovered to function as effectors of cellular transformation.
• the interaction of SMRT with PLZF-RAR ⁇ and PML-RAR ⁇ has been discovered to play a critical role in the pathogenesis of acute promyelocytic leukemia (APL) .
• APL acute promyelocytic leukemia
• Figure 1 (A) provides a schematic representation showing an alignment of SMRT and N-CoR and the boundaries of N- and C-terminal deletion mutants. Hatched boxes indicate the previously identified repressor domains (RD1 and RD2; see Horlein et al . , Nature 377:397-404 (1995)) and the checkered boxes indicate the location of receptor interaction domains (ID1 and 2; Seol et al . , Mol. Endo. 10:1646-1655 (1996). Arrows indicate the location of bacterial protease digestion sites in SMRT.
• the GAL4-DNA binding domain (DBD) 1-147 was fused to the N-terminus of these constructs and increasing amounts (0.02, 0.1, 0.5 mg) were tested in transient transfection assays for repressor activity (% of the basal activity in the presence of GAL4-DBD only) .
• the minimal repressor domains of SMRT (SRD-1 and SRD-2) are shaded. Repression values of 10 fold or higher are boxed.
• Figure 1(B) illustrates the interaction of SMRT with mSin3A in yeast . Mean values of at least 6 independent measurements are presented. Also illustrated is a schematic representation of mSin3A with amphipathic helix (PAH) domains (1-4) shown as boxes.
• PAH amphipathic helix
• Figure 1 (C) diagrams the interaction of mSin3A with SMRT and N-CoR.
• Figure 2 (A) shows that DNA bound HDAC1 (HDAC- GAL4) is a potent repressor of transcriptional activation, resulting in a 60 fold repression of basal activity.
• Figure 2 (B) shows the relief of HDAC1 dependent repression by VP-SMRT 38-811.
• Figure 2(C) illustrates that full length SMRT (but not SMRT 982-1495) squelches the relieving effect of VP-SMRT 38-811 on HDAC I dependent transcriptional repression.
• Figure 3 (A) shows the potentiation of 9-cis retinoic acid (9-cis RA) induced differentiation by the histone deacetylase inhibitor Trichostatin A (TSA) .
• TSA histone deacetylase inhibitor
• CD14 expression levels of HL-60 cells treated with the indicated amount of Trichostatin A (TSA) , 9-cis RA alone or in combination were determined by flow cytometry.
• the mean fluorescence intensities (FL2) from a representative experiment are presented.
• Figure 3 (B) illustrates CDllb expression levels on HL-60 cells treated with the indicated amount of TSA or 9-cis RA alone or in combination.
• Figure 3 diagrams hormonal targeting of nuclear complexes to chromatin template.
• a SMRT, mSin3A and HDAC1 complex associates with unliganded receptor heterodimers .
• histone deacetylase activity creates a repressed chromatin environment .
• Figure 4 (A) depicts the interaction between SMRT and PLZF in a mammalian two-hybrid assay in CV1 cells.
• the TK-MHIOO-Luc reporter used has four GAL4 binding sites .
• Figure 4 (B) shows the transcriptional repression by GAL-PLZF and GAL-PLZF-RAR ⁇ in CV1 cells.
• FIG. 5 provides a schematic representation of SMRT domain structures and the mutants used: "SRD-1" and “SRD-2” refer to SMRT repression domains 1 and 2, respectively; “IDI” and “IDII”, refer to nuclear receptor interaction domains I and II, respectively; PIO_ and PID 2 , refer to N- and C-terminal PLZF (promyelocytic leukemia zinc finger) interaction domains, respectively. Mutants I to V were used for in vi tro translation.
• Figure 6 (A) provides a schematic representation of the chromosomal translocations that generate PLZF-RAR ⁇ and PML-RAR ⁇ (promyelocytic leukemia - RAR ⁇ ) .
• FIG. 6 illustrates activation of GAL-PLZF-RAR ⁇ by histone deacetylase inhibitors Trichostatin A (TSA) (150 nM) or sodium butyrate (NaB) (5 mM) synergized with all-trans retinoic acid (ATRA) .
• TSA trichostatin A
• NaB sodium butyrate
• ATRA all-trans retinoic acid
• Figure 6(C) illustrates the relief by NaB (5 mM) of transcriptional inhibition by PLZF-RAR ⁇ and PML-RAR ⁇ on TK- ⁇ RAREx2-Luc reporters in the presence and absence of 100 nM ATRA, respectively.
• Figure 7 (A) provides a comparison of ATRA dose-dependent interaction between SMRT and RAR ⁇ or
• Figure 7(B) shows the effects of RA on TSA potentiated differentiation of NB4 cells.
• the Y-axis indicates the percentage of NBT-positive cells after treatments with ATRA and/or TSA for three days.
• Figure 7(C) shows CD18 induction after treatments with 9-cis RA and/or TSA for three days as observed by representative flow cytometry analysis. No expression was detected in control cells.
• Figure 8 shows the level of differentiation of the retinoid-resistant NB4-R4 cells induced by the combination of TSA and ATRA. The results are based on
• NBT-positive cells after treatments with ATRA and/or TSA for three days.
• a histone deacetylase inhibitor effective to modulate said hormone mediated process.
• a subject with a neoplastic disease comprising administering to the subject an amount of an inhibitor of a co-repressor, preferably inhibitors of histone deacetylase, effective to ameliorate the symptoms of said neoplastic disease.
• an inhibitor of a co-repressor preferably inhibitors of histone deacetylase
• Exemplary neoplastic diseases contemplated for treatment employing invention methods include cancer of the breast, bladder, ovary, lung and colon, Burkitt's, astrocytoma, Kaposi's sarcoma, lymphoma, leukemia, and the like.
• the present method can be employed for the treatment of subjects afflicted with leukemia, including CML and acute leukemia
• Inhibitors contemplated for inclusion in the above-described composition include histone deacetylase inhibitors (e.g., Trichostatin A (TSA), Trapoxin, sodium butyrate (NaB) , and the like) , chromatin remodeling machinery inhibitors, and the like.
• histone deacetylase inhibitors e.g., Trichostatin A (TSA), Trapoxin, sodium butyrate (NaB) , and the like
• TSA Trichostatin A
• NaB sodium butyrate
• chromatin remodeling machinery inhibitors e.g., chromatin remodeling machinery inhibitors, and the like.
• oncogene peptide refers to the protein product of an oncogene, or fragments thereof, capable of inducing one or more characteristics of cancer cells.
• exemplary oncogenes include sis, hst, erb B, fms, mas, abl , ras, mos, myc, fos, j un , pml , plzf,
• invention methods can be employed to relieve repression caused by oncogene peptides which contain the
• BTB Bric-a-brac/Tramtrack/Broad Complex
• oncogene peptides include PLZF, LAZ3/BCL6, PML, and the like.
• the oncogene peptide is an oncogenic fusion protein wherein the oncogene product, or fragment thereof, is operatively associated with a member of the steroid/thyroid hormone superfamily of receptors.
• oncogenic fusion proteins include PML- RAR ⁇ and PLZF-RAR ⁇ .
• PLZF-RAR ⁇ binds retinoic acids (RA) with affinity similar to RAR.
• RA retinoic acids
• inhibition of SMRT-mediated repression restores retinoid responsiveness to PLZF-RAR ⁇ . Based on this observation, it is believed that ligand-insensitive association of SMRT with the PLZF moiety of PLZF-RAR ⁇ underlies the retinoid non-responsiveness.
• modulate refers to the ability of a modulator for a member of the steroid/thyroid superfamily to either directly (by binding to the receptor as a ligand) or indirectly (as a precursor for a ligand or an inducer which promotes production of ligand from a precursor) induce expression of gene(s) maintained under hormone expression control, or to repress expression of gene(s) maintained under such control.
• hormone mediated processes refers to biological, physiological, endocrinological, and other bodily processes which are mediated by receptor or receptor combinations which are responsive to the ligands described herein. Modulation of such processes can be accomplished in vi tro or in vivo . In vivo modulation can be carried out in a wide range of subjects, such as, for example, humans, rodents, sheep, pigs, cows, and the like. Exemplary processes contemplated to be modulated include neoplastic diseases, inflammatory or infectious diseases, and the like.
• biological system refers to an intact organism or a cell-based system containing the various components required for response to the ligands described herein, e.g., an isoform of RAR (i.e., RAR ⁇ , RAR ⁇ or RAR ⁇ ) , a silent partner for the RAR isoform (e.g., RXR) , and an RAR-responsive reporter (which typically comprises an RAR response element (RARE) in operative communication with a reporter gene; suitable reporters include luciferase, chloramphenicol transferase, ⁇ -galactosidase, and the like.
• RAR isoform of RAR
• RXR a silent partner for the RAR isoform
• RARE RAR response element
• Contacting in a biological system contemplated by the present invention can be accomplished in a variety of ways, and the treating agents contemplated for use herein can be administered in a variety of forms (e.g., in combination with a pharmaceutically acceptable carrier therefor) and by a variety of modes of delivery.
• exemplary pharmaceutically acceptable carriers include carriers suitable for oral, intravenous, subcutaneous, intramuscular, intracutaneous, and the like administration. Administration in the form of creams, lotions, tablets, dispersible powders, granules, syrups, elixirs, sterile aqueous or non-aqueous solutions, suspensions or emulsions, and the like, is contemplated.
• suitable carriers include emulsions, solutions, suspensions, syrups, and the like, optionally containing additives such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents, and the like.
• suitable carriers include sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
• non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
• Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized, for example, by filtration through a bacteria-retaining filter, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile water, or some other sterile injectable medium immediately before use.
• the phrase "effective amount” refers to levels of compound sufficient to provide circulating concentrations high enough to modulate the expression of gene(s) mediated by members of the steroid/thyroid superfamily of receptors. Such a concentration typically falls in the range of about 10 nM up to 2 ⁇ M; with concentrations in the range of about 100 nM up to 500 nM being preferred. Since the activity of different compounds described herein may vary considerably, and since individual subjects may present a wide variation in severity of symptoms, it is up to the practitioner to determine a subject's response to treatment and vary the dosages accordingly.
• histone deacetylase enzymes are known in the art, any of which are contemplated for use in the practice of the present invention, including HDAC1, Rpd3, and the like.
• a ligand for a member of the steroid/thyroid superfamily of receptors is administered to said system in addition to said histone deacetylase inhibitor.
• the ligand can be administered independent of the histone deacetylase inhibitor or concurrently with the histone deacetylase inhibitor.
• the ligand and the histone deacetylase inhibitor are administered concurrently as part of invention composition (s) , as described herein.
• ligand for a member of the steroid/thyroid hormone superfamily of receptors
• ligand precursor for a member of the steroid/thyroid hormone superfamily of receptors
• intracellular receptor refers to a substance or compound which, in its unmodified form (or after conversion to its "active" form) , inside a cell, binds to receptor protein, thereby creating a ligand/receptor complex, which in turn can activate an appropriate hormone response element.
• a ligand therefore is a compound which acts to modulate gene transcription for a gene maintained under the control of a hormone response element, and includes compounds such as hormones, growth substances, non-hormone compounds that modulate growth, and the like.
• Ligands include steroid or steroidlike compounds, retinoids, thyroid hormones, pharmaceutically active compounds, and the like. Individual ligands may have the ability to bind to multiple receptors.
• the phrase "members of the nuclear receptor superfamily” refers to hormone binding proteins, or functional fragments thereof, that operate as ligand-dependent transcription factors, including identified members of the steroid/thyroid superfamily of receptors for which specific ligands have not yet been identified (referred to hereinafter as "orphan receptors"). These hormone binding proteins have the intrinsic ability to bind to specific DNA sequences. Following binding, the transcriptional activity of target gene (i.e., a gene associated with the specific DNA sequence) is modulated as a function of the ligand bound to the receptor.
• target gene i.e., a gene associated with the specific DNA sequence
• DNA-binding domains of all of these nuclear receptors are related, consisting of 66-68 amino acid residues, and possessing about 20 invariant amino acid residues, including nine cysteines.
• a member of the superfamily can be identified as a protein which contains the above-mentioned invariant amino acid residues, which are part of the DNA-binding domain of such known steroid receptors as the human glucocorticoid receptor (amino acids 421-486) , the estrogen receptor (amino acids 185-250) , the mineralocorticoid receptor (amino acids 603-668), the human retinoic acid receptor (amino acids 88-153) .
• the highly conserved amino acids of the DNA-binding domain of members of the superfamily are as follows:
• X designates non-conserved amino acids within the DNA-binding domain; the amino acid residues denoted with an asterisk are residues that are almost universally conserved, but for which variations have been found in some identified hormone receptors; and the residues enclosed in parenthesis are optional residues (thus, the DNA-binding domain is a minimum of 66 amino acids in length, but can contain several additional residues) .
• Exemplary members of the steroid/thyroid superfamily of receptors include steroid receptors such as glucocorticoid receptor, mineralocorticoid receptor, progesterone receptor, androgen receptor, vitamin D 3 receptor, and the like; plus retinoid receptors, such as RAR ⁇ , RAR ⁇ , RAR ⁇ , and the like, plus RXR ⁇ , RXR ⁇ , RXR ⁇ , and the like; thyroid receptors, such as TR ⁇ , TR ⁇ , and the like; as well as other gene products which, by their structure and properties, are considered to be members of the superfamily, as defined hereinabove.
• steroid receptors such as glucocorticoid receptor, mineralocorticoid receptor, progesterone receptor, androgen receptor, vitamin D 3 receptor, and the like
• retinoid receptors such as RAR ⁇ , RAR ⁇ , RAR ⁇ , and the like, plus RXR ⁇ , RXR ⁇ , RXR ⁇ , and the like
• thyroid receptors such
• orphan receptors examples include the PPARs (e.g., PPAR ⁇ , PPAR ⁇ and PPAR ⁇ ) , HNF4 [see, for example, Sladek et al., in Genes & Development 4:2353-2365 (1990)], the COUP family of receptors [see, for example, Miyajima et al., in Nucleic Acids Research 16:11057-11074 (1988), Wang et al., in Na ture 340:163-166 (1989)], COUP-like receptors and COUP homologs, such as those described by Mlodzik et al., in Cell 60:211-224 (1990) and Ladias et al., in Science 251:561-565 (1991), the ultraspiracle receptor [see, for example, Oro et al., in Na ture 347:298-301 (1990)], and the like.
• PPARs e.g., PPAR ⁇ , PPAR ⁇ and
• the retinoic acid receptor (RAR) , the thyroid hormone receptor (T 3 R) , the vitamin D 3 receptor (VDR) and the fatty acid/peroxisome proliferator activated receptor (PPAR) preferentially bind to DNA as heterodimers with a common partner, the retinoid X (or 9- cis retinoic acid) receptor (RXR; see, for example, Yu et al., in Cell 67:1251-1266 (1991); Bugge et al., in EMBO J.
• compositions comprising:
• ligands contemplated for inclusion in the above-described compositions are ligands for retinoid receptors (e.g., all-trans retinoic acid, 9-cis retinoic acid, and the like) , ligands for thyroid hormone receptors (e.g., thyroid hormone), ligands for vitamin D 3 receptor (e.g., 1, 25-dihydroxy vitamin D) , and the like.
• retinoid receptors e.g., all-trans retinoic acid, 9-cis retinoic acid, and the like
• thyroid hormone receptors e.g., thyroid hormone
• vitamin D 3 receptor e.g., 1, 25-dihydroxy vitamin D
• isolated co- repressor complexes comprising:
• the phrase "isolated” refers to peptides which have been removed from their native environment, either by enrichment thereof from natural sources, by chemical synthesis, by recombinant production, and the like.
• the recombinant expression of the above-described co-repressor complex would produce an "isolated" protein complex, since such expression would produce the peptides in a non-native environment.
• substantial enrichment of the co-repressor complex content of a cell extract would also provide an "isolated" peptide complex.
• Co-repressors contemplated by the above- described complexes include co-repressor (s) having a structure and function characteristic of SMRT (i.e., s_ilencing mediator for etinoic acid and thyroid receptors), repressor domains of SMRT (e.g., SRD-1, SRD-2, amino acids 1-981 thereof, and the like) , oncogene interaction domains of SMRT (e.g., PLZF interaction domains encompassing amino acids 467-660 ( PID-iJ and 1,174- 1,330 (PID 2 ) ) , mSin3A, protein-protein interaction domains of mSin3A (e.g., PAH-1, PAH-2, PAH-3, PAH-4, combinations of PAH, and the like) , N-CoR, Mad/Mxi-1, mSin3B, Sin3, histone deacetylase enzymes, functional fragments of any of the above, and the like, as well as combinations
• histone deacetylase enzymes known in the art, any of which can be included in the above-described complexes, e.g., HDAC1, Rpd3, and the like.
• the co-repressor complex is a critical component of switches which control cell cycle regulation and cancer.
• co-repressor complexes function as integrators in multiple transcriptional regulatory pathways to control cell growth and differentiation.
• Transcriptional co-repressors such as SMRT and N-CoR associate with non-liganded receptors resulting in suppression of basal transcriptional activity (Chen and Evans Nature 377:454-457 (1995); Chen et al. PNAS 93:7567- 7571 1996; Horlein et al. Na ture 377:397-404 (1995); Sande and Privalsky Mol Endo 10:813-825 (1996).
• mSin3A associates with Mad/Mxi-1 :Max heterodimers to promote differentiation (Ayer et al . Cell 80:767-776
• compounds are contemplated which promote dissociation of the co-repressor complex from hormone receptors (e.g., retinoid and/or thyroid hormone receptors) and further promote association of co-repressor complexes with Mad/Mxi-1 growth inhibitors.
• hormone receptors e.g., retinoid and/or thyroid hormone receptors
• homodimer/heterodimer refers to a homodimeric or heterodimeric form of one or more members of the steroid/thyroid hormone superfamily of receptors, wherein at least one of said members contains a silencing domain which represses basal level promoter activity of target genes.
• Homodimeric or heterodimeric members of the steroid/thyroid hormone superfamily of receptors contemplated for use herein include thyroid hormone receptor homodimer, thyroid hormone receptor- retinoid X receptor heterodimer, retinoic acid receptor homodimer, retinoic acid receptor-retinoid X receptor heterodimer, retinoid X receptor homodimer, and the like.
• said method comprising:
• DNA binding domain (or, in an alternative embodiment, an activation domain) , operatively associated with at least one co-repressor, a second fusion protein comprising an activation domain (or, in an alternative embodiment, a GAL4 DNA binding domain) , operatively associated with a histone deacetylase, and a reporter construct comprising a GAL4 response element operatively linked to a reporter gene; and (b) selecting those test compounds which cause reduced expression of the reporter gene product.
• the term "disrupt” embraces compounds which cause substantially complete dissociation of the various components of the complex, as well as compounds which merely alter the conformation of one or more components of the complex so as to reduce the repression otherwise caused thereby.
• cells contemplated for use in the practice of the present invention include transformed cells, non-transformed cells, neoplastic cells, primary cultures of different cell types, and the like.
• Exemplary cells which can be employed in the practice of the present invention include Schneider cells, CV-1 cells, HuTu80 cells, F9 cells, NTERA2 cells, NB4 cells, HL-60 cells, 293 cells, Hela cells, yeast cells, and the like.
• Preferred host cells for use in the functional bioassay system are COS cells and CV-1 cells.
• COS-1 (referred to as COS) cells are monkey kidney cells that express SV40 T antigen (Tag) ; while CV-1 cells do not express SV40 Tag.
• Tag SV40 T antigen
• CV-1 cells are presently preferred because they are particularly convenient for gene transfer studies and provide a sensitive and well-described host cell system.
• physiological conditions are readily understood by those of skill in the art to comprise an isotonic, aqueous nutrient medium at a temperature of about 37°C.
• fusion proteins comprising an activation domain or a GAL4 DNA binding domain operatively associated with any one of the fusion protein components described herein.
• exemplary fusion proteins comprise an activation domain operatively associated with a histone deacetylase, a co-repressor, an oncogene peptide, and the like, as well as a GAL4 DNA binding domain operatively associated with a histone deacetylase, a co-repressor, an oncogene peptide, and the like.
• the GAL4 DNA binding domain of the yeast GAL4 protein comprises at least the first 74 amino acids thereof (see, for example, Keegan et al., Science 231:699-704 (1986)).
• the first 90 or more amino acids of the GAL4 protein will be used, with the first 147 amino acid residues of yeast GAL4 being presently most preferred.
• Activation domains contemplated for use in the practice of the present invention are well known in the art and can readily be identified by the artisan. Examples include the GAL4 activation domain, BP64, VP16, and the like.
• Exemplary GAL4 response elements are those containing the palindromic 17-mer:
• reporter genes include chloramphenicol transferase (CAT) , luciferase (LUC) , beta-galactosidase ( ⁇ -gal) , and the like.
• the phrase "operatively associated with” means that the respective DNA sequences, or the polypeptides encoded thereby (represented, for example, by the terms “GAL4 response element” and “reporter gene) are operational, i.e., work for their intended purposes; the word “functionally” means that after the two segments are linked, upon appropriate activation by a ligand-receptor complex, the reporter gene will be expressed as the result of the fact that the corresponding "response element" was "turned on” or otherwise activated.
• Two or more polypeptides are operatively associated or linked if each polypeptide component works for its intended purpose and still retains biological function, (e.g., binding, co-repression, activation, and the like) .
• the above-described assay can be modified to facilitate identification of compounds which disrupt any of the specific interactions involved in the formation of the above-described complex.
• modified host cell comprises: a first fusion protein comprising an activation domain, operatively associated with at least one co-repressor, a second fusion protein comprising a GAL4 DNA binding domain, operatively associated with a histone deacetylase, a first reporter construct comprising a GAL4 response element operatively linked to a first reporter gene, and a second reporter construct comprising a hormone response element operatively linked to a second reporter gene; and
• HREs hormone response elements
• Naturally occurring HREs are composed of direct repeats (i.e., DRs; see Umesono et al., in Cell 65:1255- 1266 (1991), inverted repeats (i.e., IRs; see Umesono et al., in Na ture 336:262-265 (1988), and Williams et al. in J. Biol . Chem . 266:19636-19644 (1991)), and/or everted repeats (ERs; see Baniahmad et al., in Cell 61:505-514 (1990); Farsetti et al., in J. Biol . Chem . 267:15784-15788 (1992); Raisher et al., in J.
• direct repeats i.e., DRs
• IRs inverted repeats
• IRs see Umesono et al., in Na ture 336:262-265 (1988)
• ERs see Baniahmad et
• the X n sequence also serves as a gap which separates the two core-binding sites.
• spacers of 1, 3, 4 and 5 nucleotides serve as preferred response elements for heterodimers of RXR with PPAR, VDR, T 3 R and RAR, respectively (see, for example, Naar et al., in Cell 65:1267-1279 (1991); Umesono et al., 1991, supra; Kliewer et al., in Na ture 358:771-774 (1992); and Issemann et al., supra) .
• the optimal gap length for each heterodimer is determined by protein-protein contacts which appropriately position the DNA binding domains (DBDs) of RXR and its partner (see, for example, Kurokawa et al. Genes Dev. 7:1423-1435 (1993); Perlmann et al. Genes Dev. 7:1411-1422 (1993); Towers et al.P roc. Na tl . Acad. Sci . USA 90:6310-6314 (1993); and Zechel et al. EMBO J. 13:1414-1424 (1994) ) .
• DBDs DNA binding domains
• Direct repeat hormone response elements contemplated for use in the practice of the present invention are composed of at least one direct repeat of two or more half sites, optionally separated by one or more spacer nucleotides (with spacers of 1-5 preferred) .
• the spacer nucleotides can be selected from any one of A, C, G or T.
• Each half site of direct repeat HREs contemplated for use in the practice of the invention comprises the sequence
• R is selected from A or G; B is selected from G, C, or T; each N is independently selected from
• M is selected from A or C; with the proviso that at least 4 nucleotides of said -RGBNNM- sequence are identical with the nucleotides at corresponding positions of the sequence -AGGTCA- .
• Response elements employed in the practice of the present invention can optionally be preceded by N x , wherein x falls in the range of 0 up to 5.
• said method comprising:
• modified host cell comprises: a first fusion protein comprising a
• GAL4 DNA binding domain (or, in an alternative an activation domain) , operatively associated with at least one co-repressor, a second fusion protein comprising an activation domain (or, in an alternative a GAL4 DNA binding domain) , operatively associated with a histone deacetylase, and a reporter construct comprising a GAL4 response element operatively linked to a reporter gene;
• test compounds which prevent ligand-induced reduction of expression of the reporter gene product.
• a wide variety of compounds can be assayed employing the invention method. Any compound with the potential to act as a ligand can be tested, e.g., steroid or steroidlike compounds, retinoids, thyroid hormones, pharmaceutically active compounds, naturally occurring compounds, synthetic organic compounds, and the like.
• said method comprising:
• affinity matrix comprises: an affinity support, a first fusion protein comprising a member of the steroid/thyroid hormone superfamily of receptors, operatively associated with a glutathione-S- methionine (GST) label (or, in an alternative embodiment, a HIS label) , a second fusion protein comprising a heterologous partner for said member, operatively associated with a HIS label (or, in an alternative embodiment, a GST label) , and at least one co-repressor; and
• modified host cell comprises: a first fusion protein comprising a GAL4 DNA binding domain (or, in an alternative embodiment, an activation domain) , operatively associated with at least one co-repressor, a second fusion protein comprising an activation domain (or, in an alternative embodiment, a GAL4 DNA binding domain) , operatively associated with said oncogene peptide, and a reporter construct comprising a GAL4 response element operatively linked to a reporter gene; and
• said method comprising: (a) contacting an affinity matrix with a test compound, wherein said affinity matrix comprises: an affinity support, a first fusion protein comprising a co-repressor, operatively associated with a glutathione-S- methionine (GST) label (or, in an alternative embodiment, a HIS label) , a second fusion protein comprising an oncogene peptide, operatively associated with a HIS label (or, in an alternative embodiment, a GST label) , and at least one co-repressor; and
• CMX-SMRT CMX-C-SMRT
• CMX-N-SMRT CMXGAL-C-SMRT
• CMXGAL-C-SMRT Cho-Set al. (1995) Nature 377: 454-457
• CMX-PML-RAR ⁇ Kakizuka et al . (1991) Cell 66:663-674
• CMXGAL-mSin3A PAH1-4; Nagy et al . (1997) Cell 89:373-380
• pSG5-PLZF and pSG5-PLZF-RAR ⁇ Choen et al . (1994) Proc . Natl Acad. Sci . USA 91:1178-1182
• pSG5-PML-RAR ⁇ m4 ; Shao et al . (1997) Blood 89:4282-4289
• HA-RAR ⁇ amino acids
• HA-PLZF and HA-PLZF-RAR ⁇ were constructed by PCR to incorporate a HA tag at the 5 ' end of corresponding cDNA sequence and cloned into pCMX.
• SMRT deletion mutants and GST fusions were cloned into pCMX and pGEX-KG respectively.
• VP-PML-RAR ⁇ was generated by fusion of corresponding cDNA sequence to VP16-AD in CMX-VP16.
• CMX-hRAR ⁇ 411 was constructed by introduction of a stop codon at amino acid 411 of human RAR ⁇ (Robertson et al . (1992) Blood 80 : 1885-1889) using Quick-change mutagenesis kit ( ⁇ tratagene) . All PCR derived products were verified by sequencing.
• the anti-SMRT polyclonal antibody used for immunoblotting was produced in a rabbit injected with purified GST-SMRT (amino acids 38-448) proteins. Rabbit anti-SMRT antibody used for immunofluorescence has been described (Chen et al . (1996) Proc . Natl Acad Sci . USA.
• Mouse anti-HA (12CA5) antibody was purchased from Boehringer Mannheim.
• Goat AP-conjugated secondary antibodies were purchased from BioRad. Donkey
• Histone deacetylase inhibitors were obtained from CalBiochem (sodium butyrate) and Waco Pure Chemical
• Example 2 SMRT has two independent repressor domains
• the silencing activity of SMRT resides in the N- terminal half (amino acids 1-981) of the protein, while the receptor interaction domain (ID) is in the remaining C-terminal segment (Chen and Evans, Nature 377:454-457 (1995)).
• Minimal, transferable repressor domain(s) were identified in order to understand the mechanism of transcriptional repression and its molecular basis.
• SMRT-GAL4 constructs were generated by PCR amplification of the indicated regions and fused to GAL4 DNA binding domain (DBD) 1-147
• Repressor activity was determined by transiently transfecting into CV-1 cells increasing amounts of the GAL4-fusion vectors along with a reporter construct pMH- 100 TK-luc which contains 4 GAL4 binding sites. (Chakravarti et al . , Nature 383:99-103 (1996)). Fold repression was determined relative to the basal transcriptional activity of the reporter in the presence of GAL4 DBD alone. Luciferase activity of each sample was normalized by the level of ⁇ -galactosidase activity. Each transfection was carried out in triplicate and repeated 3- 6 times. Yeast transformation and ⁇ -galactosidase activity assays were carried out in strain Y190 according to manufacturers protocol (Clonetech) . The results of this assay are illustrated in Figure 1A.
• SMRT 38-811 appears to be as potent a repressor (45 fold) as either full length SMRT (35 fold) or SMRT 1- 981 (30 fold) , suggesting that in fact it contains all the domains necessary for full repression. Additional nested C-terminal deletions revealed a smaller though less potent repressor domain, SMRT 38-448 (12 fold) . Further C- terminal deletions significantly lowered (38-370, 2.8 fold) and abolished (38-266) repressor activity. N- terminal deletions of SMRT 38-448 revealed that the minimal repressor domain resides between amino acids 259- 448 (12-fold repression) . Further deletions abolished the repressor activity (364-448). Thus, amino acids 259-448 define an autonomous S.MRT xepressor domain (SRD-1) .
• SRD-1 autonomous S.MRT xepressor domain
• SRD-1 is a structural domain is supported by the observation that there were several sites susceptible to protease digestion by bacterial proteases in the vicinity of the boundaries of SRD-1 (see Figure 1A arrows) . Expanding SRD-1 towards the C-terminus (259-811) yielded a construct with increased repressor activity (100-fold) suggesting the presence of a second repressor domain. Additional deletions localized the boundary of a second, autonomous minimal repressor domain between amino acids 559-657 (50 fold repression) which is termed SRD-2. SRD-1 and SRD-2 share substantial homology with the comparable region in N-CoR (42% and 39%, respectively) suggesting functional conservation.
• the GAL4 activation domain (AD) gives a low background reporter activity.
• GAL4 DBD GAL4 DNA binding domain
• GAL4-SMRT GAL4-SMRT
• association with mSin3A was mapped to two regions of SMRT, amino acids 259-448, which correspond to SRD-1 and amino acids 449-657, which corresponds to SRD-2, respectively. Consistent with the domain mapping, further deletions (38- 214, 38-266, and 336-370) completely abolish association with mSin3A. Therefore, these results suggest that the repressor activities of SRD1 and SRD2 are mediated via association with mSin3A.
• Amino acid sequence 1-192 contains amphipathic helix (PAH) domain 1 (PAH1) ; 1-386 contains both PAH domains 1 and 2; 1-529 contains PAH domains 1, 2, and 3; and 1-965 contains all four PAH domains plus the conserved linker between PAH 3 and 4.
• PAH amphipathic helix
• N-CoR The SMRT-related co-repressor, N-CoR, was also examined to determine if it associates with mSin3A via SRD-1 and SRD-2 related regions. While the boundaries of these regions have not been determined in detail, results indicate that N-CoR also interacts with mSin3A.
• GST fusion proteins of 38-266, 38-448 and 548-811 of SMRT were examined for their ability to bind in vi tro translated S-Methionine-labeled mSin3A in pull down experiments.
• GST-SMRT 38-266, 38-448 and 548-811 were purified from E. coli cells and extracts were passed through a Glutathione Sepharose 4B affinity column
• mSin3A and B were compared for their ability to interact with SRD-1.
• S Methionine labeled mSin3A, mSin3B and PAH domains of mSin3A (PAHl (112-192) , PAH1-2 (112-386) , PAH1-3 (112-529) , PAH1-4 (112-965) were used as probes in GST pull down experiments as described above with GST-SMRT 38-448 (S) or GST (G) ) .
• S GST-SMRT 38-448
• GST (G) GST
• Radiolabeled GAL4-DBD fusion of PAH1-4 of mSin3A was incubated either with GST or GST-RXR LBD/6 His-RAR LBD heterodimer and labeled SMRT in the absence or presence of 5 TM all-trans retinoic acid (atRA) for 2h at 4°C. Bound proteins were eluted with IX SDS PAGE buffer and separated on a 7.5 or 12.5 % SDS-PAGE. Gels were fixed, dried and exposed to film. Both mSin3A (PAHl-4) and SMRT are retained on the matrix in the absence of ligand and are released in a retinoic acid dependent fashion.
• HDAC1 is a mediator of SMRT silencing
• direct recruitment of HDAC1 to a heterologous promoter should result in repression of basal activity.
• This prediction was tested by fusing HDAC1 to the GAL4 DBD and assaying its effect on the basal activity of the GAL4- dependent reporter in transient transfection assays in CV- 1 cells.
• the reporter gene contained GAL4 binding sites upstream of a minimal TK promoter fused to luciferase gene (pMHl00-TK-luc) . Normalized luciferase activity was determined and fold repression (relative to GAL4-DBD alone) was calculated.
• FIG. 2A shows that HDACl-GAL4 is a potent repressor of transcriptional activation resulting in a 60-fold repression of basal activity. Similar results were recently reported by Yang, W-M. et al . , Nature 382:319-324 (1996) using mammalian homologs of Rpd3.
• transfection of increasing amounts of full length SMRT displaces the VP- SMRT activator and re-establishes repression to an approximately 50% level (lane 6) .
• co- transfection of the carboxy terminal domain of SMRT fails to squelch the VP-SMRT/HDAC1 interaction.
• TSA Trichostatin A
• TSA 100 nM TSA showed a minimal effect on the CD14 marker while a suboptimal dose (10 nM) of 9-cis RA resulted in modest stimulation (Fig. 3A) .
• a suboptimal dose (10 nM) of 9-cis RA resulted in modest stimulation (Fig. 3A) .
• addition of both TSA and lOnM 9-cis RA resulted in dramatic enhancement of CD14 expression to levels higher than that following lOOnM 9-cis RA treatment.
• the high dose 9-cis RA treatment was also enhanced by TSA.
• Similar results were seen with the CDllb marker, although in this case low doses of TSA partially activated gene expression (Fig. 3B) .
• the combination of TSA and 9-cis RA proved to be cooperative at both high and low doses.
• Recombinant proteins were obtained as follows and in vi tro protein interaction assays were carried out as follows.
• GST-PLZF and GST-SMRT fusion proteins were produced from E. Coli DH5a or BL-21 cells and purified by glutathione-Sepharose 4B affinity chromatography. Bound proteins were eluted with 15 mM glutathione. Purified proteins were then reloaded on glutathione-Sepharose 4B beads and used as affinity matrices. Protein interaction assays were performed in G buffer (20 mM Tris, 7.9, 150 mM KC1, 1 mM EDTA, 4 mM MgCl 2 , 0.2% NP-40, 10% Glycerol) at 4°C for 60 min with gentle rocking. The beads were washed five times with G buffer and bounded proteins were eluted in SDS sample buffer, separated by SDS-PAGE and visualized by autoradiography.
• GST Ras-RAR ⁇ or GST-PLZF (amino acids 7-118) affinity matrices and analyzed by SDS-PAGE together with 20% of the input.
• GST-PLZF amino acids 7-118 affinity matrices and analyzed by SDS-PAGE together with 20% of the input.
• BTB (Bric-a-brac/Tramtrack/Broad Complex) domain of PLZF (GST-PLZF)
• GST-RAR ⁇ retained only C-SMRT as expected (Chen et al. (1995) Nature 377:454-457) .
• the association of PLZF with SMRT in intact cells was demonstrated using a mammalian two-hybrid assay.
• SMRT was examined to determine whether it localizes to the same structures.
• HA-tagged PLZF or PLZF-RAR ⁇ and SMRT were expressed in 293T cells and their subcellular localization was determined by immunohistochemical techniques .
• vi tro immunoprecipitation in vi tro translated proteins were incubated together with 10 mg/ml anti-HA antibody in G buffer at 4°C for 60 min. Immunocomplexes were isolated by incubation with protein A-agarose, washed four times with G buffer, analyzed by SDS-PAGE and visualized by autoradiography.
• 2x10 CVl cells were transfected with 10 mg of appropriate plasmids and harvested in ice-cold PBS 24 hours after transfection.
• IP buffer 10 mM NaF, 25 mM b-glycerolphosphate, 0.1% NP-40, 0.5 mM PMSF plus protease inhibitors
• Immunoprecipitation was performed at 4°C overnight in the presence and absence of 5mM ATRA. Immunocomplexes were resolved by SDS-PAGE and analyzed by immunoblotting. Immunohistochemistry was done as previously described (Dyck et al . Cell 76:333-343 (1994). PLZF and PLZF-RAR ⁇ were detected by the mouse anti-HA antibody following instructions from the manufacturer (Boehringer Mannheim) .
• SMRT or N-CoR associates with mSin3 and histone deacetylases (HDACs) to mediate transcriptional repression by nuclear receptors and Mad/Max complex (Nagy et al . (1997) Cell 89:373-380; Heinzel et al . (1997) Nature 387:43-48; Alland et al . (1997) Nature 387:49-55).
• HDACs histone deacetylases
• anti-HA immunoprecipitation was performed using in vi tro translated HA-PLZF, SMRT, mSin3A and HDAC-1 proteins .
• Radiolabelled SMRT, mSin3A and HDAC-1 were co-immunoprecipitated with PLZF.
• the PLZF/SMRT complex associated with mSin3A (PAH 1-4) and HDAC-1, suggesting they formed a quaternary complex.
• the observation that SMRT interacts with PLZF, together with the observation that SMRT interacts with LAZ3/BCL6, provide the first evidence that oncogenes containing the BTB domain utilize the same repression pathway as nuclear receptors and Mad/Max.
• this domain alone, which has been shown to be required for the oncogenic function of PLZF-RAR ⁇ (Horlein et al . (1995) Nature 377:397-404; Ruthardt et al . (1997) Mole . Cell . Biol . 17:4859-4869), is sufficient to interact with SMRT and recruit the histone deacetylase complex.
• PML-RAR ⁇ and PLZF-RAR ⁇ are the two major oncogenic fusion proteins identified in APL patients (Fig. 6 (A) ) .
• Affinity matrices were employed to determine whether 35 S-labelled RAR ⁇ or PLZF-RAR ⁇ would interact with immobolized GST-SMRT-IDII (amino acids 1,073-1,168) and GST-SMRT-PID 2 protein in the presence or absence of 5 mM ATRA.
• GAL4-DBD fusions of RAR ⁇ or PLZF-RAR ⁇ were transfected into CVl cells and examination of their RA-activation on UAS reporters.
• CVl cells were transfected using DOTAP transfection reagents (Boehringer Mannheim) . Luciferase activity of each sample was normalized against internal control ⁇ -galactosidase activity (Nagy et al . (1997) Cell 89:373-380). Each transfection was carried out in triplicate and repeated at least three times. When indicated, transfected cells were treated with ATRA alone for 36 hours or 16 hours with ATRA plus histone deacetylase inhibitors.
• ATRA compared to GAL-RAR ⁇ (Fig. 6(B), columns A and B) , presumably due to the association with SMRT.
• treatment with histone deacetylase inhibitors Trichostatin A (TSA) or Na-butyrate (NaB) which inhibit SMRT-mediated repression (Nagy et al . (1997) Cell 89:373-380; Heinzel et al. (1997) Nature 387:43-48), relieved repression by GAL-PLZF-RAR ⁇ and synergized with ATRA to activate reporters (columns C and D) . It was then sought to determine whether similar effects can be seen on native RA-response elements.
• histone deacetylase inhibitor NaB was able to restore nearly full RA activation in the presence of PLZF-RAR ⁇ .
• NaB also partially relieves transcriptional inhibition by PML-RAR ⁇ , suggesting that SMRT and histone deacetylases mediates the oncogenic function of PML-RAR ⁇ as well.
• PML-RAR ⁇ oncogene blocks myeloid differentiation in vi tro and in vivo (Grignani et al . (1993) Cell 74:423-431; Brown et al . (1997) Proc . Natl Acad. Sci . USA. 94:2551-2556). In contrast to PLZF-RAR ⁇ , however, this block can be overcome by high doses of RA. It has been proposed that PML-RAR ⁇ causes leukemia by interfering with either the RAR or PML dependent differentiation pathways (Dyck et al . (1994) Cell 76:333-343; Weis et al . (1994) Cell 76:345-356; Grignani et al .
• PML to RAR ⁇ generates a receptor with significantly decreased ability to release SMRT upon ligand binding, suggesting that PML modulates the interaction between SMRT and the RAR moiety through an allosteric mechanism since the RA-binding affinity of PML-RAR ⁇ appears to be normal (Chen et al . (1994) Proc . Natl Acad . Sci . USA 91:1178-1182; Ruthardt et al . (1997) Mole . Cell . Biol . 17:4859-4869) and PML does not physically interact with SMRT, unlike PLZF (Chen et al . (1995) Nature 377:454-457).
• PML-RAR ⁇ fusion proteins Brown et al . (1997) PNAS 94:2551-2556; Grignani et al . , (1994) Blood 83:10-25
• PML-RAR ⁇ could serve as RA-dependent activators.
• introduction of PML-RAR ⁇ into non-responsive hematopoietic cells renders them sensitive to supra-physiological concentrations of retinoids ( Grignani et al .
• NB4 cell line established from a patient with t(15;17) APL.
• NB4, NB4-R4 Shao et al . (1997) Blood 89:4282-4289), HL-60 and HL-60R (Robertson et al . (1992) Blood 80:1885-1889) cells were cultured in RPMI with 10% FBS.
• Differentiation effects of ATRA and/or TSA on those cells were assessed using the Nitroblue Tetrazolium (NBT) reduction assay.
• NBT assays with RA and/or TSA were carried out as previously described (Nagy et al . (1997) Cell 89:373-380). NBT assay has been described (Nagy et al . (1995) Mole . Cell . Biol . 15:3540-3551) .
• CD18 For flow cytometry analysis of CD18 expression, the cells were incubated with a FITC-conjugated anti-CD18 antibody (DAKO) in a dilution of 1:500 in PBS (0.5% BSA) followed by extensive washing before analyzing on a FACSCAN flow cytometer (Becton Dickinson) .
• DAKO FITC-conjugated anti-CD18 antibody
• PBS 0.5% BSA
• FACSCAN flow cytometer Becton Dickinson
• Resistance to differentiation by physiological concentrations of RA is a general feature of APL. Although t(15;17) patients initially respond to high doses of retinoid, many of them relapse and develop permanent resistance (Grignani et al . (1994) Blood 83:10-25). Moreover, acquired retinoid resistance in otherwise sensitive HL-60 and NB4 cell lines (HL-60R and NB4-R4) has been traced to dominant negative mutations in RAR ⁇ (RAR ⁇ 411) and PML-RAR ⁇ (m4) respectively, that reduce RA binding (Robertson et al . (1992) Blood 801885-1889; Shao et al . (1997) Blood 89:4282-4289).

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