Human histone deacetylase, HDAC9c Number:7,521,044 from the United States Patent and Trademark Office (PTO) owispatent The present invention relates to newly discovered human histone deacetylases (HDACs), also referred to as histone deacetylase-like polypeptides. The polynucleotide sequences and encoded polypeptides of the novel HDACs are encompassed by the invention, as well as vectors comprising these polynucleotides and host cells comprising these vectors. The invention also relates to antibodies that bind to the disclosed HDAC polypeptides, and methods employing these antibodies. Also related are methods of screening for modulators, such as inhibitors or antagonists, or agonists. The invention also relates to diagnostic and therapeutic applications which employ the disclosed HDAC polynucleotides, polypeptides, and antibodies, and HDAC modulators. Such applications can be used with diseases and disorders associated with abnormal cell growth or proliferation, cell differentiation, and cell survival, e.g., neoplastic cell growth, and especially breast and prostate cancers or tumors.<H deacetylase, histone, Human, histone, d, 7521044.html, Patent, pto, 7521044

Title: Human histone deacetylase, HDAC9c

Abstract: The present invention relates to newly discovered human histone deacetylases (HDACs), also referred to as histone deacetylase-like polypeptides. The polynucleotide sequences and encoded polypeptides of the novel HDACs are encompassed by the invention, as well as vectors comprising these polynucleotides and host cells comprising these vectors. The invention also relates to antibodies that bind to the disclosed HDAC polypeptides, and methods employing these antibodies. Also related are methods of screening for modulators, such as inhibitors or antagonists, or agonists. The invention also relates to diagnostic and therapeutic applications which employ the disclosed HDAC polynucleotides, polypeptides, and antibodies, and HDAC modulators. Such applications can be used with diseases and disorders associated with abnormal cell growth or proliferation, cell differentiation, and cell survival, e.g., neoplastic cell growth, and especially breast and prostate cancers or tumors.

Patent Number: 7,521,044 Issued on 04/21/2009 to Jackson, et al.

Inventors: Jackson; Donald G. (Lawrenceville, NJ), Lorenzi; Matthew V. (Philadelphia, PA), Lee; Liana M. (San Francisco, CA), Feder; John N. (Belle Mead, NJ)
Assignee: Bristol-Myers Squibb Company (Princeton, NJ)

Appl. No.: 11/497,205

Filed: August 1, 2006

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
10172094	Jun., 2002	7199227
60298296	Jun., 2001

Current U.S. Class: 424/94.1 ; 435/183; 530/350

Current International Class: A61K 38/43 (20060101); C07K 14/00 (20060101); C12N 9/00 (20060101)

References Cited [Referenced By]

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4218478	August 1980	Omura et al.
6068987	May 2000	Dulski et al.
6110697	August 2000	Dulski et al.
6600351	July 2003	Bisanti et al.
7063973	June 2006	Richon et al.
2003/0129724	July 2003	Grozinger et al.

Foreign Patent Documents


1394274	Mar., 2004	EP
WO95/11994	May., 1995	WO
WO95/25116	Sep., 1995	WO
WO02/102984	Dec., 2002	WO
WO03/029451	Apr., 2003	WO
WO03/065006	Aug., 2003	WO
WO03/068268	Aug., 2003	WO
WO03/087768	Oct., 2003	WO
WO2004/005513	Jan., 2004	WO
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Primary Examiner: Goddard; Laura B
Attorney, Agent or Firm: D'Amico; Stephen C.

Parent Case Text

RELATED APPLICATIONS

This application is a divisional application of non-provisional application U.S. Ser. No. 10/172,094, filed Jun. 14, 2002, now U.S. Pat. No. 7,199,227, which claims benefit to provisional application U.S. application Ser. No. 60/298,296, filed Jun. 14, 2001, which is incorporated by reference in its entirety.

Claims

What is claimed is:

1. An isolated polypeptide comprising a polypeptide sequence selected from the group consisting of: (a) an isolated polypeptide comprising amino acids 1 to 1069 of SEQ ID NO :87; (b) an isolated polypeptide comprising amino acids 2 to 1069 of SEQ ID NO:87; (c) an isolated polypeptide comprising the polypeptide sequence encoded by nucleotides 1 to 3207 of SEQ ID NO:88; and (d) an isolated polypeptide comprising the polypeptide sequence encoded by nucleotides 4 to 3207 of SEQ ID NO:88.

2. The isolated polypeptide of claim 1, wherein said polypeptide is (a).

3. The isolated polypeptide of claim 1, wherein said polypeptide is (b).

4. The isolated polypeptide of claim 1, wherein said polypeptide is (c).

5. The isolated polypeptide of claim 1, wherein said polypeptide is (d).

6. An isolated polypeptide produced by a method comprising: (a) culturing an isolated recombinant host cell comprising a vector that comprises the coding region encoding the polypeptide of claim 1 under conditions such that said polypeptide is expressed; and (b) recovering said polypeptide.

7. The isolated polypeptide of claim 1 further comprising a heterologous polypeptide sequence.

8. An isolated polypeptide comprising at least 920 contiguous amino acids of SEQ ID NO:87.

9. An isolated polypeptide encoded by cDNA clone, HDAC9c, contained in ATCC Deposit No: PTA-4454.

Description

FIELD OF THE INVENTION

The present invention relates to novel members of the histone deacetylase (HDAC) family, including BMY_HDAL1, BMY_HDAL2, BMY_HDAL3, BMY_HDACX_v1, BMY_HDACX_v2, and HDAC9c. Specifically related are nucleic acids encoding the polypeptide sequences, vectors comprising the nucleic acid sequences, and antibodies that bind to the encoded polypeptides. In addition, the invention relates to pharmaceutical compositions and diagnostic reagents comprising one or more of the disclosed HDAC components. The present invention also relates to methods of treating a disease or disorder caused by malfunction of an HDAC, e.g., due to mutation or altered gene expression. The invention further relates to methods of using a modulator of an HDAC of the present invention to treat or ameliorate a disease state. Also related are methods for devising antisense therapies and prophylactic treatments using the HDACs of the invention. In particular, the disclosed HDAC components and methods may be used to prevent, diagnose, and treat diseases and disorders associated with abnormal cell growth or proliferation, cell differentiation, or cell survival, e.g., neoplasias, cancers, and tumors, such as breast and prostate cancers or tumors, and neurodegerative diseases.

BACKGROUND OF THE INVENTION

Chromatin is a dynamic protein-DNA complex which is modulated by post-translational modifications. These modifications, in turn, regulate cellular processes such as gene transcription and replication. Key chromatin modifications include the acetylation and deacetylation of nucelosomal histone proteins. Acetylation is catalyzed by histone acetylases (HATs), whereas deacetylation is catalyzed by deacetylases (HDACs or HDAs). HDACs catalyze the removal of acetyl groups from the N-termini of histone core proteins to produce more negatively charged chromatin. This results in chromatin compaction, which shuts down gene transcription. In addition, inhibition of HDACs results in the accumulation of hyperacetylated histones. This, in turn, is implicated in a variety of cellular responses, including altered gene expression, cell differentiation, and cell-cycle arrest (see, generally, S. G. Gray et al., 2001, Exp. Cell Res. 262(2):75-83, and U.S. Pat. Nos. 6,110,697 and 6,068,987 to Dulski et al.).

The HDAC gene family is composed of two distinct classes. Class I HDACs are related to the yeast transcriptional regulator, RPD3. Class II HDACs include a subgroup of proteins containing a C-terminal catalytic domain as well as a separate N-terminal domain with transcriptional repression activity. Class III HDAC proteins are related to the yeast sir2 protein and require NAD for activity. Class I HDACs are predominantly nuclear, whereas class II HDACs are transported between the cytoplasm and nucleus as part of the regulation of cellular proliferation and/or differentiation (reviewed in S. Khochbin et al., 2001, Curr. Opin. Genet Dev. 11(2):162-6).

The best characterized substrates for HDACs include histone or histone-like peptide sequences containing N-terminal lysines. However, non-histone HDAC substrates have also been identified, including several transcription factors. Non-histone substrates for HDACs include p53, androgen receptor, LEF1/TCF4 (B. R. Henderson et al., 2002, J. Biol. Chem., published online on May 1, 2002 as Manuscript M110602200), GATA-1, and estrogen receptor-alpha (reviewed in D. M. Vigushin et al., 2002, Anticancer Drugs 13(1):1-13). For these substrates, deacetylation has been shown to regulate DNA/protein interactions or protein stability. Such molecules may therefore represent therapeutic targets of HDACs. Importantly, the histone deacetylase function of HDACs represses transcription by removing the acetyl moieties from amino terminal lysines on histones, thereby resulting in a compact chromatin structure. In contrast, the non-histone deacetylase function of HDACs can either repress or activate transcription.

There has been considerable interest in modulating the activity of HDACs for the treatment of a variety of diseases, particularly cancer. Several small molecule inhibitors of HDAC have shown anti-proliferative activities on a number of tumor cell lines and potent anti-tumor activity in pre-clinical tumor xenograft models, most recently, CBHA (D. C. Coffey et al., 2001, Cancer Res. 61(9):3591-4), pyroxamide, (L. M. Butler et al, 2001, Clin. Cancer Res. 7(4):962-70), and CHAP31 (Y. Komatsu et al., 2001, Cancer Res. 61(11):4459-66). Several inhibitors are presently being evaluated as single agents and in combination regimens with cytotoxic agents for the treatment of advanced malignancies (reviewed in P. A. Marks et al., Curr. Opin. Oncol. 2001 November; 13(6):477-83). Thus, HDAC inhibitors are being developed as anti-tumor agents, as well as agents useful for gene therapy (McInerney et al., 2000, Gene Ther. 7(8):653-663).

Small molecule inhibitors of HDAC activity that have undergone extensive analysis include trichostatin A (TSA), trapoxin, SAHA (V. M. Richon et al., 2001, Blood Cells Mol. Dis. 27(1):260-4), CHAPs (Y. Komatsu et al., 2001, Cancer Res. 61(11):4459-66), MS-27-275 (reviewed in M. Yoshida et al., 2001, Cancer Chemother. Pharmacol. 48 Suppl. 1:S20-6), depsipeptide (FR901228; FK228; see, e.g., V. Sandor et al., 2002, Clin. Cancer Res. 8(3):718-28), and CI-994 (see, e.g., P. M. LoRusso et al., 1996, New Drugs 14(4):349-56; S. Prakash et al., 2001, Invest. New Drugs 19(1):1-11). Trichostatin A and trapoxin have been reported to be reversible and irreversible inhibitors, respectively, of mammalian histone deacetylase (Yoshida et al, 1995, Bioassays, 17(5):423-430). Trichostatin A has also been reported to inhibit partially purified yeast histone deacetylase (Sanchez del Pino et al., 1994, Biochem. J., 303:723-729). Moreover, trichostatin A is an antifungal antibiotic and has been shown to have anti-trichomonal activity and cell differentiating activity in murine erythroleukemia cells, as well as the ability to induce phenotypic reversion in ras-transformed fibroblast cells (see e.g. U.S. Pat. No. 4,218,478; and Yoshida et al., 1995, Bioassays, 17(5):423-430, and references cited therein). Trapoxin A, a cyclic tetrapeptide, induces morphological reversion of v-sis-transformed NIH/3T3 cells (Yoshida and Sugita, 1992, Jap. J. Cancer Res., 83(4):324-328).

The therapeutic effects of HDAC inhibition are believed to occur through the induction of differentiation and/or apoptosis through the up-regulation of genes such as the cyclin dependent kinase inhibitors, p21 and p27 (see, e.g., W. Wharton et al., 2000, J. Biol. Chem. 275(43):33981-7; L. Huang et al., 2000, Mol. Med. 6(10):849-66). Although known HDAC inhibitors are efficacious as anti-tumor agents, they are also associated with toxicity (see, e.g., V. Sandor et al., 2002, Clin. Cancer Res. 8(3):718-28). Such toxicity is believed to be caused by a non-selective mechanism of targeting multiple HDACs. Despite the potent anti-tumor activity of HDAC inhibitors, it is still unclear which HDACs are necessary to produce an anti-proliferative response. Furthermore, little progress has been made in comparing the HDAC gene expression profiles in tumor versus normal cells. Differential HDAC expression may underlie the tumor-selective responses of HDAC inhibition. In addition, a cellular growth advantage may be conferred by the expression of particular HDACs. Therefore, there is a need for further insight into the consequences of selective HDAC inhibition, or activation.

SUMMARY OF THE INVENTION

The present invention provides novel histone deacetylase (HDAC) nucleic acid sequences and their encoded polypeptide products, also called histone deacetylase like (HDAL) sequences and products herein, as well as methods and reagents for modulating HDACs.

It is an aspect of this invention to provide new HDAC nucleic acid or protein sequences, or cell lines overexpressing HDAC nucleic acid and/or encoded protein, for use in assays to identify small molecules which modulate HDAC activity, preferably antagonize HDAC activity.

It is another aspect of the present invention to employ HDAC protein structural data for the in silico identification of small molecules which modulate HDAC activity. This structural data could be generated by experimental techniques (for example, X-Ray crystallography or NMR spectroscopy) or by computational modeling based on available histone deacetylase structures (for example, M. S. Finnin et al., 1999, Nature, 401(6749):188-193).

Another aspect of the present invention provides modulators of HDAC activity, e.g., antagonists or inhibitors, and their use to treat neoplastic cells, e.g., cancer cells and tumor cells. In one aspect of the invention, breast or prostate cancers or tumors are treated using the HDAC modulators. The modulators of the invention can be employed alone or in combination with standard anti-cancer regimens for neoplastic cell, e.g., tumor and cancer, treatments.

In addition, the present invention provides diagnostic reagents (i.e., biomarkers) for the detection of cancers, tumors, or neoplastic growth. In one embodiment, HDAC (e.g., HDAC9c) nucleic acids or anti-HDAC antibodies are used to detect the presence of specific cancers or tumors, such as breast or prostate cancers or tumors.

It is yet another aspect of the present invention to employ HDAC inhibitors in the regulation of the differentiation state of normal cells such as hematopoietic stem cells. According to this invention, a method is provided for the use of modulators of HDAC in ex vivo therapies, particularly as a means to modulate the expression of gene therapeutic vectors.

Yet another aspect of this invention is to provide antisense nucleic acids and oligonucleotides for use in the regulation of HDAC and HDAL gene transcription or translation.

An additional aspect of this invention pertains to the use of HDAC nucleic acid sequences and antibodies directed against the produced protein for prognosis or susceptibility for certain disorders (e.g., breast or prostate cancer).

Further aspects, features and advantages of the present invention will be better appreciated upon a reading of the detailed description of the invention when considered in connection with the accompanying figures/drawings.

BRIEF DESCRIPTION OF THE FIGURES

The file of this patent contains at least one figure executed in color. Copies of this patent with color figure(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

FIG. 1 shows the novel BMY_HDAL1 partial nucleic acid (cDNA) sequence (SEQ ID NO:1) and the encoded amino acid sequence (SEQ ID NO:2) of the BMY_HDAL1 polypeptide product. The top line in each group of FIG. 1 presents the BMY_HDAL1 protein sequence (SEQ ID NO:2) in 3-letter IUPAC form; the middle line presents the nucleotide sequence of the BMY_HDAL1 coding strand (i.e., SEQ ID NO:1); and the bottom line presents the nucleotide sequence of the reverse strand (SEQ ID NO:3).

FIGS. 2A and 2B show the amino acid sequences of the novel histone deacetylase-like proteins BMY_HDAL1 (SEQ ID NO:2), BMY_HDAL2 (SEQ ID NO:4) and BMY_HDAL3 (SEQ ID NO:5) aligned with the following known histone deacetylase proteins: S. cerevisiae HDA1 (SC_HDA1), (SEQ ID NO:6); human HDAC4 (HDA4), (SEQ ID NO:7); human HDAC5 (HDA5), (SEQ ID NO:8); human HDAC7 (HDA7), (SEQ ID NO:9) and to a histone deacetylase-like protein ACUC from Aquifex aeolicus (AQUIFEX_HDAL), (SEQ ID NO:10), (M. S. Finnin et al., 1999, Nature, 401(6749):188-193). Residues identical among all proteins are in shown in black text on a gray background. The sequences were aligned using the ClustalW algorithm as implemented in the VectorNTI sequence analysis package (1998, 5.5 Ed., Informax, Inc.) with a gap opening penalty of 10, a gap extension penalty of 0.1 and no end gap penalties.

FIGS. 3A and 3B show a GenewiseDB comparison of BMY_HDAL1 amino acid sequence (SEQ ID NO:2) and human HDAC5 (HDA5) amino acid sequence (SEQ ID NO:8). Genewise results from HDA5_HUMAN_run2 applied to AC002088 nucleic acid (coding) sequence. (SEQ ID NO:11).

FIG. 4 presents the results of sequence motif analysis of motifs within the BMY_HDAL1 amino acid sequence.

FIG. 5 shows the novel BMY_HDAL2 partial nucleic acid (cDNA) sequence (SEQ ID NO:12) and the encoded amino acid sequence (SEQ ID NO:4) of the BMY_HDAL2 polypeptide product. The top line in each group of FIG. 5 presents the BMY_HDAL2 protein sequence (SEQ ID NO:4) in 3-letter IUPAC form; the middle line presents the nucleotide sequence of the BMY_HDAL2 coding strand (i.e., SEQ ID NO:12); and the bottom line presents the nucleotide sequence of the reverse strand (SEQ ID NO:13).

FIG. 6 presents a GenewiseDB comparison of the BMY_HDAL2 amino acid sequence (SEQ ID NO:4) and human HDAC5 (HDA5) amino acid sequence (SEQ ID NO:8). Genewise results from HDA5_HUMAN_run3 applied to AC002410 nucleic acid sequence (SEQ ID NO:14).

FIG. 7 shows PROSITE motifs identified in the predicted amino acid sequence of the novel BMY_HDAL2 (SEQ ID NO:4). MOTIFS are from: bmy_hdal2.aa.fasta.

FIGS. 8A and 8B show the sequences of the N- and C-terminal sequences of BMY_HDAL3 as determined from BAC AC004994 and BAC AC004744. FIG. 8A presents the most N-terminal region of the BMY_HDAL3 amino acid sequence (SEQ ID NO:15) presented herein as encoded by the human genomic BAC AC004994 polynucleotide sequence (SEQ ID NO:17). FIG. 8B presents an additional C-terminal portion of the BMY_HDAL3 amino acid sequence (SEQ ID NO:16) as encoded by human genomic BAC AC004744 polynucleotide sequence (SEQ ID NO:18).

FIG. 9 shows partial transcripts identified from the AC004994 polynucleotide sequence (SEQ ID NO:17) and from the AC004744 polynucleotide sequence (SEQ ID NO:18) assembled into a single contig, which was designated BMY_HDAL3 (SEQ ID NO:19) using the VectorNTI ContigExpress program (Informax, Inc.).

FIG. 10 presents the BMY_HDAL3 partial nucleic acid sequence (SEQ ID NO:19) and the encoded amino acid sequence (SEQ ID NO:5) based on the assembled BMY_HDAL3 sequence described in FIG. 9. The top line in each group of FIG. 10 presents the BMY_HDAL3 protein sequence (SEQ ID NO:5) in 3-letter IUPAC form; the middle line presents the nucleotide sequence of the BMY_HDAL3 coding strand (i.e., SEQ ID NO:19); and the bottom line presents the nucleotide sequence of the reverse strand (SEQ ID NO:20).

FIG. 11 presents the results of the GCG Motifs program used to analyze the BMY_HDAL3 partial predicted amino acid sequence for motifs in the PROSITE collection (K. Hofmann et al., 1999, Nucleic Acids Res., 27(1):215-219) with no allowed mismatches.

FIG. 12 shows a multiple sequence alignment of the novel human HDAC, BMY_HDAL3, amino acid sequence (SEQ ID NO:5) with the amino acid sequence of AAC78618 (SEQ ID NO:21) and with the amino acid sequence of AAD15364 (SEQ ID NO:22). AA78618 is a histone deacetylase-like protein predicted by genefinding and conceptual translation of AC004994 and which was entered in Genbank. AAD15364 is a similar predicted protein derived from AC004744 and entered in Genbank. AAC78618, AAD15364 and BMY_HDAL3 were aligned using the ClustalW algorithm as implemented in the VectorNTI sequence analysis package (1998, 5.5 Ed., Informax, Inc.) with a gap opening penalty of 10, a gap extension penalty of 0.1 and no end gap penalties. Residues identical among all proteins are shown in white text on a black background; conserved residues are shown in black text on a gray background.

FIG. 13 shows a BLASTN alignment of the AA287983 polynucleotide sequence (SEQ ID NO:23) and BMY_HDAL3 polynucleotide sequence from SEQ ID NO:19. Genbank accession AA287983 is a human EST sequence (GI # 1933807; Incyte template 1080282.1) which was identified by BLASTN searches against the Incyte LifeSeq database using the NCBI Blast algorithm (S. F. Altschul et al., 1997, Nucl. Acids Res., 25(17):3389-3402) with default parameters. The AA287983 human EST was isolated from a germinal B-cell library. No additional ESTs are included in the Incyte template derived from this cluster (Incyte gene ID 180282).

FIGS. 14A-14H present other histone deacetylase sequences, as shown in FIGS. 2A and 2B. FIG. 14A: Aquifex ACUC protein amino acid sequence (SEQ ID NO:10); FIG. 14B: Saccharomyces cerevisiae histone deacetylase 1 amino acid sequence (SEQ ID NO:6); FIG. 14C: Homo sapiens histone deacetylase 4 amino acid sequence (SEQ ID NO:7); FIG. 14D: Homo sapiens histone deacetylase 5 amino acid sequence (SEQ ID NO:8); FIG. 14E: Homo sapiens histone deacetylase 7 amino acid sequence (SEQ ID NO:9); FIG. 14F: Human EST AA287983 nucleic acid sequence (SEQ ID NO:23); FIG. 14G: Human predicted protein AAD15364 amino acid sequence (SEQ ID NO:22); and FIG. 14H: Human predicted protein AAC78618 amino acid sequence (SEQ ID NO:21).

FIGS. 15A-15C depict the nucleotide and amino acid sequence information for HDAC9c. The polypeptide sequence (SEQ ID NO:87) is shown using the standard 3-letter abbreviation for amino acids. The DNA sequence (SEQ ID NO:88) of the coding strand is also shown. FIGS. 15D-15F depict an amino acid sequence alignment of HDAC9c. The predicted amino acid sequence of HDAC9c (SEQ ID NO:87) was aligned to previously identified HDACs, including HDAC9 (AY032737; SEQ ID NO:89), HDAC9a (AY032738; SEQ ID NO:90), and HDAC4 (ALF132608; SEQ ID NO:91), using ClustalW (D. G. Higgins et al., 1996, Methods Enzymol. 266:383-402). Identical amino acids are shown in white text on a black background; conserved amino acids are shown in black text on a gray background.

FIGS. 16A-16C depict expression levels of HDAC9 and HDAC9c in human cancer cell lines and normal adult tissue. FIG 16A: Northern blot analysis of HDAC9 and HDAC9c expression in normal adult tissue. FIG 16B: Quantitative PCR mRNA analysis of HDAC9 and HDAC9c expression in human tumor cell lines. FIG 16C: Nuclease protection assay analysis of HDAC9 and HDAC9c expression in human tumor cell lines. FIG. 16D shows the nucleotide sequence of HDAC9c used to derive the probes used for Northern blotting and nuclease protection analysis (SEQ ID NO:92). The probes were derived from the HDAC9c nucleotide sequence, and were predicted to hybridize to HDAC9c and HDAC9 (AY032737), but not HDAC9a (AY032738).

FIGS. 17A-17C illustrate the increase of HDAC9 and HDAC9c gene expression in human cancer tissues. FIGS. 17A-17B: Summary of HDAC9 and HDAC9c expression in selected tissues, as assayed by in situ hybridization. FIG. 17C: Photomicrographs of representative cells showing HDAC9 and HDAC9c or actin staining.

FIG. 18 shows HDAC9c-mediated induction of morphological transformation of NIH/3T3 cells. The panels show photomicrographs of soft agar growth of vector (upper panel), FGF8 (middle panel) and HDAC9c (lower panel) transfected NIH/3T3 cells. Cells are shown at 10.times. magnification.

FIG. 19 shows HDAC9c induction of actin stress fiber formation in NIH/3T3 cells. Stable NIH/3T3 cells expressing the indicated constructs were stained with phalloidin-TRITC and visualized by fluorescent microscopy.

FIGS. 20A-20C depict the nucleotide and amino acid sequence information for BMY_HDACX variant 1, also called BMY_HDACX_v1 and HDACX_v1. BMY_HDACX_v1 represents a partial cDNA sequence obtained from cells expressing a transcript variant of human HDAC9. The polypeptide sequence (SEQ ID NO:93) is shown using the standard 3-letter abbreviation for amino acids. The DNA sequence (SEQ ID NO:94) of the coding strand is also shown.

FIGS. 21A-21B depict the nucleotide and amino acid sequence information for BMY_HDACX variant 2, also called BMY_HDACX_v2 and HDACX_v2. BMY_HDACX_v2 represents a full-length sequence of a novel transcript variant (i.e., splice product) of HDAC9. The polypeptide sequence (SEQ ID NO:95) is shown using the standard 3-letter abbreviation for amino acids. The DNA sequence (SEQ ID NO:96) of the coding strand is also shown.

FIGS. 22A-22I depict the nucleotide and amino acid sequence information for the previously identified HDAC9 transcript variants. FIGS. 22A-22C: HDAC9 variant 1 (HDAC9v1; NCBI Ref. Seq. NM.sub.--058176). The polypeptide sequence (SEQ ID NO:89) is shown using the standard 3-letter abbreviation for amino acids. The DNA sequence (SEQ ID NO:97) of the coding strand is also shown. FIGS. 22D-22F: HDAC9 variant 2 (HDAC9v2; NCBI Ref. Seq. NM.sub.--058177). The polypeptide sequence (SEQ ID NO:90) is shown using the standard 3-letter abbreviation for amino acids. The DNA sequence (SEQ ID NO:98) of the coding strand is also shown. FIGS. 22G-22I: HDAC9 variant 3 (HDAC9v3; NCBI Ref. Seq. NM.sub.--014707). The polypeptide sequence (SEQ ID NO:99) is shown using the standard 3-letter abbreviation for amino acids. The DNA sequence (SEQ ID NO:100) of the coding strand is also shown.

FIGS. 23A-23K depict a multiple sequence alignment of nucleotide sequences representing known and novel HDAC9 splice products. The cDNAs for BMY_HDACX-v1 (SEQ ID NO:94) and BMY_HDACX_v2 (SEQ ID NO:96) nucleotide sequences were aligned to the three reported splice products of the HDAC9 gene, including HDAC9v1 (NCBI Ref. Seq. NM.sub.--058176; SEQ ID NO:97), HDAC9v2 (NCBI Ref. Seq. NM.sub.--058177; SEQ ID NO:98), and HDAC9v3 (NCBI Ref. Seq. NM.sub.--014707; SEQ ID NO:100) using the sequence alignment program ClustalW (D. G. Higgins et al., 1996, Methods Enzymol. 266:383-402). The consensus sequence is shown on the bottom line (SEQ ID NO:106). Identical nucleotides are shown in white text on a black background. Selected splice junctions are indicated below the alignment; these junctions were identified by comparison of the cDNA sequences to the assembled genomic contig NT.sub.--00798.1 using the Sim4 algorithm (L. Florea et al., 1998, Genome Res. 8:967-74). It is noted that the HDAC9 (AY032737) nucleotide and amino acid sequences are identical to the HDAC9v1 (NM.sub.--058176) nucleotide and amino acid sequences. Similarly, the HDAC9a (AY032738) nucleotide and amino acid sequences are identical to the HDAC9v2 (NM.sub.--058177) nucleotide and amino acid sequences.

FIGS. 24A-24D depict a multiple sequence alignment of amino acid sequences representing known and novel HDAC polypeptides. The amino acid sequences encoded by transcript variants BMY_HDACX_v1 (SEQ ID NO:93) and BMY_HDACX v2 (SEQ ID NO:95) were aligned to amino acid sequences encoded by known splice variants of human histone deacetylase 9 including HDAC9v1 (NCBI Ref. Seq. NM.sub.--058176; SEQ ID NO:89), HDAC9v2 (NCBI Ref Seq. NM.sub.--058177; SEQ ID NO:90), and HDAC9v3 (NCBI Ref. Seq. NM.sub.--014707; SEQ ID NO:99), and to human histone deacetylases 4 and 5 (HDA5, SEQ ID NO:8; HDA4, SEQ ID NO:7) using the multiple sequence alignment program ClustalW (D. G. Higgins et al., 1996, Methods Enzymol. 266:383-402). The consensus sequence is shown on the bottom line (SEQ ID NO:107). Residues conserved among all polypeptides are shown in white text on a black background; residues conserved in a majority of polypeptides are shown in black text on a gray background.

FIGS. 25A-25C depict a multiple sequence alignment of amino acid sequences showing novel HDAC polypeptides. The amino acid sequences of BMY_HDAL1 (SEQ ID NO:2), BMY_HDAL2 (SEQ ID NO:4), BMY_HDAL3 (SEQ ID NO:5), HDAC9c (SEQ ID NO:87), HDACX_v1 (SEQ ID NO:93), and HDACX_v2 (SEQ ID NO:95) were aligned using the T-Coffee program (C. Notredame et al., 2000, J. Mol. Biol. 302:205-217; C. Notredame et al., 1998, Bioinformatics 14:407-422). Identical residues are shown in black text on a gray background.

DESCRIPTION OF THE INVENTION

The present invention discloses several novel HDAC nucleotide sequences and encoded products. New members of the histone deacetylase protein family have been identified as having identity to known HDACs. Three new HDACs are referred to as BMY_HDAL1, BMY_HDAL2, and BMY_HDAL3 herein, wherein HDAL signifies histone deacetylase like proteins in current nomenclature. These proteins are most similar to the known human histone deacetylase, HDAC9. Novel HDAC9 splice variants, termed HDACX_v1 and HDACX_v2, have also been identified. In addition, HDAC9c, an HDAC9-related family member, has been newly identified and cloned. The nucleic acid sequences encoding the novel HDAC polypeptides are provided together with the description of the means employed to obtain these novel molecules. Such HDAC products can serve as protein deacetylases, which are useful for disease treatment and/or diagnosis of diseases and disorders associated with cell growth or proliferation, cell differentiation, and cell survival, e.g., neoplastic cell growth, cancers, and tumors.

As shown herein, HDAC9 expression is elevated in tumor cell lines, as determined by quantitative PCR analysis. Elevated expression of HDAC9 was also observed in clinical specimens of human tumor tissue compared to normal tissue, using in situ hybridization (ISH) and an HDAC9-specific riboprobe. Further, cell biological assessment of HDAC9c revealed that overexpression of HDAC9c confers a growth advantage to normal fibroblasts. These results indicate that HDAC9c can be used as a diagnostic marker for tumor progression and that selective HDAC9c inhibitors can be used to target specific cancer or tumor types, such as breast and prostate cancers or tumors.

Definitions

The following definitions are provided to more fully describe the present invention in its various aspects. The definitions are intended to be useful for guidance and elucidation, and are not intended to limit the disclosed invention and its embodiments.

HDAC polypeptides (or proteins) refer to the amino acid sequence of isolated, and preferably substantially purified, human histone deacetylase proteins isolated as described herein. HDACs may also be obtained from any species, preferably mammalian, including mouse, rat, non-human primates, and more preferably, human; and from a variety of sources, including natural, synthetic, semi-synthetic, or recombinant. The probes and oligos described may be used in obtaining HDACs from mammals other than humans. The present invention more particularly provides six new human HDAC family members, namely, BMY_HDAL1, BMY_HDAL2, BMY_HDAL3, HDACX_v1, HDACX_v2, and HDAC9c, their polynucleotide sequences (e.g., SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:19, SEQ ID NO:88, SEQ ID NO:94, SEQ ID NO:96, and sequences complementary thereto), and encoded products (e.g., SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:87, SEQ ID NO:93, and SEQ ID NO:95).

An agonist (e.g., activator) refers to a molecule which, when bound to, or interactive with, an HDAC polypeptide, or a functional fragment thereof, increases or prolongs the duration of the effect of the HDAC polypeptide. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules that bind to and modulate the effect of an HDAC polypeptide. An antagonist (e.g., inhibitor, blocker) refers to a molecule which, when bound to, or interactive with, an HDAC polypeptide, or a functional fragment thereof, decreases or eliminates the amount or duration of the biological or immunological activity of the HDAC polypeptide. Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules that decrease, reduce or eliminate the effect and/or function of an HDAC polypeptide.

"Nucleic acid sequence", as used herein, refers to an oligonucleotide, nucleotide, or polynucleotide (e.g., DNA, cDNA, RNA), and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be single- or double-stranded, and represent the sense (coding) or antisense (non-coding) strand. By way of nonlimiting example, fragments include nucleic acid sequences that can be about 10 to 60 contiguous nucleotides in length, preferably, at least 15-60 contiguous nucleotides in length, and also preferably include fragments that are at least 70-100 contiguous nucleotides, or which are at least 1000 contiguous nucleotides or greater in length. Nucleic acids for use as probes or primers may differ in length as described herein.

In specific embodiments, HDAC polynucleotides of the present invention can comprise at least 15, 20, 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1195, 1200, 1500, 2000, 2160, 2250, 2500, 2755, or 2900 contiguous nucleotides of SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:19, SEQ ID NO:88, SEQ ID NO:94, SEQ ID NO:96, or a sequence complementary thereto. Additionally, a polynucleotide of the invention can comprise a specific region of a HDAC nucleotide sequence, e.g., a region encoding the C-terminal sequence of the HDAC polypeptide. Such polynucleotides can comprise, for example, nucleotides 3024-4467 of HDAC9c (SEQ ID NO:88), nucleotides 2156-3650 of HDACX_v1 (SEQ ID NO:94), nucleotides 1174-3391 of HDACX_v2 (SEQ ID NO:96), or portions or fragments thereof.

As specific examples, polynucleotides of the invention may comprise at least 183 contiguous nucleotides of SEQ ID NO:88; or at least 17 contiguous nucleotides of SEQ ID NO:96. As additional examples, the polynucleotides of the invention may comprise nucleotides 1 to 3207 of SEQ ID NO:88; nucleotides 1 to 2340 of SEQ ID NO:94; or nucleotides 307 to 1791 of SEQ ID NO:96. Further, the polynucleotides of the invention may comprise nucleotides 4 to 3207 of SEQ ID NO:88, wherein said nucleotides encode amino acids 2 to 1069 of SEQ ID NO:87 lacking the start methionine; or nucleotides 310 to 1791 of SEQ ID NO:96, wherein said nucleotides encode amino acids 2 to 495 of SEQ ID NO:95 lacking the start methionine. In addition, polynucleotides of the invention may comprise nucleotides 3024-3207 of SEQ ID NO:88; or nucleotides 1174-1791 of SEQ ID NO:96.

"Amino acid sequence" as used herein refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragments or portions thereof, and to naturally occurring or synthetic molecules. Amino acid sequence fragments are typically from about 4 or 5 to about 35, preferably from about 5 to about 15 or 25 amino acids in length and, optimally, retain the biological activity or function of an HDAC polypeptide. However, it will be understood that larger amino acid fragments can be used, depending on the purpose therefor, e.g., fragments of from about 15 to about 50 or 60 amino acids, or greater.

Where "amino acid sequence" is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and like terms, such as "polypeptide" or "protein" are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule. In addition, the terms HDAC polypeptide and HDAC protein are frequently used interchangeably herein to refer to the encoded product of an HDAC nucleic acid sequence of the present invention.

A variant of an HDAC polypeptide can refer to an amino acid sequence that is altered by one or more amino acids. The variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan. Minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing functional biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software.

An allele or allelic sequence is an alternative form of an HDAC nucleic acid sequence. Alleles may result from at least one mutation in the nucleic acid sequence and may yield altered mRNAs or polypeptides whose structure or function may or may not be altered. Any given gene, whether natural or recombinant, may have none, one, or many allelic forms. Common mutational changes that give rise to alleles are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.

Altered nucleic acid sequences encoding an HDAC polypeptide include nucleic acid sequences containing deletions, insertions and/or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent HDAC polypeptide. Altered nucleic acid sequences may further include polymorphisms of the polynucleotide encoding an HDAC polypeptide; such polymorphisms may or may not be readily detectable using a particular oligonucleotide probe. The encoded protein may also contain deletions, insertions, or substitutions of amino acid residues, which produce a silent change and result in a functionally equivalent HDAC protein of the present invention. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological activity or function of the HDAC protein is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine.

"Peptide nucleic acid" (PNA) refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide ("oligo") linked to a peptide backbone of amino acid residues, which terminates in lysine. PNA typically comprise oligos of at least 5 nucleotides linked to amino acid residues. These small molecules stop transcript elongation by binding to their complementary strand of nucleic acid (P. E. Nielsen et al., 1993, Anticancer Drug Des., 8:53-63). PNA may be pegylated to extend their lifespan in the cell where they preferentially bind to complementary single stranded DNA and RNA.

Oligonucleotides or oligomers refer to a nucleic acid sequence, preferably comprising contiguous nucleotides, typically of at least about 6 nucleotides to about 60 nucleotides, preferably at least about 8 to 10 nucleotides in length, more preferably at least about 12 nucleotides in length, e.g., about 15 to 35 nucleotides, or about 15 to 25 nucleotides, or about 20 to 35 nucleotides, which can be typically used, for example, as probes or primers, in PCR amplification assays, hybridization assays, or in microarrays. It will be understood that the term oligonucleotide is substantially equivalent to the terms primer, probe, or amplimer, as commonly defined in the art. It will also be appreciated by those skilled in the pertinent art that a longer oligonucleotide probe, or mixtures of probes, e.g., degenerate probes, can be used to detect longer, or more complex, nucleic acid sequences, for example, genomic DNA. In such cases, the probe may comprise at least 20-200 nucleotides, preferably, at least 30-100 nucleotides, more preferably, 50-100 nucleotides.

Amplification refers to the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction (PCR) technologies, which are well known and practiced in the art (See, D. W. Dieffenbach and G. S. Dveksler, 1995, PCR Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

Microarray is an array of distinct polynucleotides or oligonucleotides synthesized on a substrate, such as paper, nylon, or other type of membrane; filter; chip; glass slide; or any other type of suitable solid support.

The term antisense refers to nucleotide sequences, and compositions containing nucleic acid sequences, which are complementary to a specific DNA or RNA sequence. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand. Antisense (i.e., complementary) nucleic acid molecules include PNA and may be produced by any method, including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes that block either transcription or translation. The designation "negative" is sometimes used in reference to the antisense strand, and "positive" is sometimes used in reference to the sense strand.

The term consensus refers to the sequence that reflects the most common choice of base or amino acid at each position among a series of related DNA, RNA, or protein sequences. Areas of particularly good agreement often represent conserved functional domains.

A deletion refers to a change in either nucleotide or amino acid sequence and results in the absence of one or more nucleotides or amino acid residues. By contrast, an insertion (also termed "addition") refers to a change in a nucleotide or amino acid sequence that results in the addition of one or more nucleotides or amino acid residues, as compared with the naturally occurring molecule. A substitution refers to the replacement of one or more nucleotides or amino acids by different nucleotides or amino acids.

A derivative nucleic acid molecule refers to the chemical modification of a nucleic acid encoding, or complementary to, an encoded HDAC polypeptide. Such modifications include, for example, replacement of hydrogen by an alkyl, acyl, or amino group. A nucleic acid derivative encodes a polypeptide that retains the essential biological and/or functional characteristics of the natural molecule. A derivative polypeptide is one that is modified by glycosylation, pegylation, or any similar process that retains the biological and/or functional or immunological activity of the polypeptide from which it is derived.

The term "biologically active", i.e., functional, refers to a protein or polypeptide or peptide fragment thereof having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active" refers to the capability of the natural, recombinant, or synthetic HDAC, or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells, for example, to generate antibodies, and to bind with specific antibodies.

An HDAC-related protein refers to the HDAC and HADL proteins or polypeptides described herein, as well as other human homologs of these HDAC or HDAL sequences, in addition to orthologs and paralogs (homologs) of the HDAC or HADL sequences in other species, ranging from yeast to other mammals, e.g., homologous histone deacetylase. The term ortholog refers to genes or proteins that are homologs via speciation, e.g., closely related and assumed to have common descent based on structural and functional considerations. Orthologous proteins function as recognizably the same activity in different species. The term paralog refers to genes or proteins that are homologs via gene duplication, e.g., duplicated variants of a gene within a genome. (See, W. M. Fritch, 1970, Syst. Zool., 19:99-113.

It will be appreciated that, under certain circumstances, it may be advantageous to provide homologs of one of the novel HDAC polypeptides which function in a limited capacity as one of either an HDAC agonist (i.e., mimetic), or an HDAC antagonist, in