WO2008033990A2 - Compositions and methods for modulating the notch signal transduction pathway - Google Patents

Abstract

The present invention relates to a p180NACK polypeptide which binds to Notch and modulates Notch-mediated signal transduction. Nucleic acid molecules encoding the p180NACK polypeptide, antibodies that selectively bind the p180NACK polypeptide, and iRNA molecules which inhibit the expression of the p180NACK polypeptide are provided, as are methods for identifying agents which modulate the expression or activity of the p180NACK polypeptide. A method for modulating Notch-mediated signal transduction using agents of the invention is also provided.

Description

COMPOSITIONS AND METHODS FOR MODULATING THE NOTCH SIGNAL TRANSDUCTION PATHWAY

Introduction

This application claims benefit of priority to U.S. Provisional Patent Application Serial No. 60/825,773, filed September 15, 2006, the content of which is incorporated herein by reference in its entirety. This invention was made in the course of research sponsored by the National Institutes of Health (NIH Grant No. CA83736) . The U.S. government may have certain rights in this invention.

Background of the Invention

The Notch signal transduction pathway is an evolutionarily conserved mechanism to mediate fundamental decisions in stem cell maintenance and differentiation in many tissue types. For example, Notchl signaling is critically important for cell fate decisions during hematopoiesis . Several lines of evidence indicate that Notch signaling plays a direct role during many of the bifurcation points, including the transition from common lymphoid progenitor cell to mature T cell . The mammalian NOTCH gene family consists of 4 closely related members {N0TCH1-N0TCH4) . Substantial evidence now indicates that NOTCHl₁ N0TCH2 and N0TCH3 play direct roles in leukemogenesis . The Notchl locus was found to be involved in the chromosomal translocation t(7;9) (q34,-q34.3) in SUP-Tl cells derived from human T-cell Acute Lymphocytic Leukemia (T-ALL) . In this translocation, the sequence encoding most of the extracellular domain is replaced with sequence derived from the T-cell receptor β locus. This results in the expression of Notchl proteins that lack nearly the entire extracellular domain and are rendered constitutively active by intracellular Notch (N^1C) -like molecules. Although this translocation is relatively rare, the intracellular portion of Notch (N^1C) is constitutively active in nearly half of all T-ALLs, by alternative mutations m the Notch gene. Reconstitution of lethally- irradiated mice with bone marrow cells that express analogous N^1C results m the development of T-cell leukemia, which indicates that aberrant N^1C expression predisposes mice to T-cell leukemia and therefore mimics some aspects of the disease m humans.

Notch signaling is initiated after direct interaction between Notch and a Delta/Serrate/lag-2 (DSL) ligand presented on an adjacent cell. This interaction results m proteolytic processing that releases N^1C from the plasma membrane. N^1C then translocates to the nucleus where it interacts with the DNA binding protein CSL and the co- activator protein Mastermmd-likel (Mamll) to activate transcription of target genes. Evidence suggests other proteins might be associated with Notch, CSL, and Mamll m this activator complex. The mechanism by which this Notch complex regulates transcription is being actively investigated. Even though the exact mechanism by which N^1C drives tumoπgenesis is not entirely understood, it is clear that N^1C must accumulate m the nucleus and incorporate into the activator complex. Mutations m N^1C that disrupt complex formation also fail to activate transcription of target genes such as cyclm Dl. Similarly, dominant-negative alleles of MAMLl encoding only the Notch binding domain sequences disrupt formation of the complex and inhibit the growth of SUP-Tl cells. Taken together, these observations indicate that the integrity of this complex is absolutely critical for N^1C transforming activity. In addition to an uncertain mechanism of action, events downstream of Notch are also not understood. To develop novel therapeutics directed against the Notch pathway, critical components of the Notch pathway, and downstream effector molecules must be identified.

Summary of the Invention

The present invention is an isolated pl80^NACK polypeptide or fragment thereof. An isolated nucleic acid molecule encoding the pl80^NACK polypeptide, or which hybridizes to the same is also provided, as is a vector containing the nucleic acid molecule and host cell harboring said vector. While some embodiments embrace an antibody that selectively binds the pl80^NACK polypeptide or fragment, other embodiments embrace a fusion protein composed of a heterologous tag fused to the pl80^NACK polypeptide or pl80^NACK fragment.

The present invention is also a screening method for identifying an agent that modulates Notch-mediated signal transduction. The method involves contacting a pl80^NACK polypeptide or cell expressing said polypeptide with a test agent and determining whether said agent modulates the expression or activity of the pl80^NACK polypeptide thereby identifying an agent that modulates Notch-mediated signal transduction. A method for modulating Notch-mediated signal transduction using an agent identified by said screening method is also provided.

Brief Description of the Drawings

Figure 1 shows an amino acid sequence alignment of mouse pl80^NACK and homologs thereof from human (SEQ ID N0:3), rhesus monkey (SEQ ID NO: 4), canine (SEQ ID NO: 5), bovine

(SEQ ID N0:6), FLJ00269 (SEQ ID N0:7), and rat (SEQ ID

NO:8). A serine/threonine protein kinase catalytic domain is indicated (###, Figure ID) as are conserved sequence motifs (SEQ ID NO:9-19).

Figure 2 shows that pl80^NΛCK is a coactivator of Notch- dependent transcription as determined using an H1299 8xCSL reporter luciferase.

Detailed Description of the Invention

A novel protein which functions in the Notch signal transduction pathway and is critical for tumor cell survival has now been identified. This protein is a previously uncharacterized protein known as FLJ00269 or D8Ertd82e (SEQ ID NO : 7 ; GENBANK Accession No. NP_766499) . Because this protein binds to Notch and modulates Notch- mediated-CSL target gene activity, the protein was designated pl80^NACK; pl80 denoting the size of the protein and NACK for Notch Activation Complex Kinase. The pl80^NACK proteins and nucleic acids disclosed herein find application in the development of novel anti -neoplastic therapeutics with specificity to tumors harboring mutations in the Notch pathway. Examples of tumors exhibiting aberrant Notch activity include, but are not limited to, lymphoma, melanoma, and cancers of the breast, lung, colon, central nervous system and pancreas. Furthermore, manipulation of Notch activity through pl80^NACK protein is useful in regulating stem cell growth and differentiation.

Accordingly, the present invention relates to an isolated pl80^NACK nucleic acid and polypeptide encoded thereby. The invention further relates to fragments and orthologs of the pl80^NACK polypeptide, as well as anti- pl80^NACK antibodies, iRNA targeting the endogenous pl80^NACK locus, screening assays for identifying agents which modulate pl80^NACK activity, and methods for treating diseases and conditions associated with aberrant Notch activity. The term isolated, as used herein in reference to a polypeptide, peptide, nucleic acid molecule, or antibody of the invention, means a polypeptide, peptide, nucleic acid molecule, or antibody that is in a form that is relatively free from contaminating lipids, polypeptides, nucleic acids or other cellular material normally associated with the polypeptide, peptide or nucleic acid molecule in a cell. In particular embodiments, the term isolated means that the polypeptide, peptide, nucleic acid molecule, or antibody is 85%, 90%, 96%, 97%, 98%, 99%, or 100% pure.

The term pl80^NACK encompasses a polypeptide having the sequence of the murine pl80^NACK polypeptide (SEQ ID NO: 2) and is intended to include orthologs of murine pl80 NACK i . e , pl 80 NACK polypeptides found in other species, which are derived from a common ancestor and have substantial amino acid sequence identity with murine pl80^NACK (SEQ ID NO : 2 ) . Exemplary mammalian pl80^NACK orthologs are shown in Figure 1 and include polypeptides isolated from rat, rhesus monkey, bovine, canine and human. While these polypeptides have been annotated as hypothetical proteins in the prior art, the substantial amino acid sequence identity of these proteins with murine pl80^NACK indicates that they are indeed orthologs of murine pl80 NACK [see Table 1) .

TABLE 1

Accordingly, the term pl80 NACK describes polypeptides generally having an amino acid sequence with greater than about 60% identity, desirably greater than about 65% identity, more desirably greater than about 75% identity, and can be a polypeptide having greater than about 80%, 90%, 95%, 97%, or 99% amino acid sequence identity with murine pl80^NΛCK (SEQ ID NO: 2) . In particular embodiments, polypeptides having such substantial amino acid sequence identity with murine pl80^NACK include those set forth in SEQ ID N0:3, SEQ ID NO : 4 , SEQ ID NO : 5 , SEQ ID NO : 6 , and SEQ ID NO: 8.

As used herein, the term "substantial amino acid sequence identity" when used in reference to a pl80^NACK amino acid sequence, is intended to mean the pl80^NACK sequence shown in Figure 1, or a similar, non-identical sequence that is considered by those skilled in the art to be a functionally equivalent amino acid sequence. For example, an amino acid sequence that has substantial amino acid sequence identity with pl80^NΛCK can have one or more modifications such as amino acid additions, deletions or substitutions relative to the amino acid sequence of pl80^NACK

(SEQ ID NO:2), provided that the modified polypeptide retains substantially at least one biological activity of pl80^NACK, such as the ability to bind to Notch and modulate Notch-mediated target gene activity. Comparison of sequences for substantial amino acid sequence identity can be performed between two sequences of any length and usually is performed with amino acid sequences of between 6 and 1400 amino acid residues. Such comparisons for substantial amino acid sequence identity are performed using methodology routine in the art, e.g., using algorithms such as CLUSTALW or DIALIGN, and are useful for identifying regions which can be modified without affecting activity.

By way of illustration, amino acid sequence comparisons between murine pl80^NACK and its orthologs identified conserved amino acid sequence motifs (SEQ ID NO: 9-19) which may be essential to the function of pl80^NACK. In particular, it is believed that the serine/threonine protein kinase catalytic domain depicted in Figure ID is essential to the activity of pl80^NACK. Therefore, it is contemplated that one or more modifications in the non- conserved regions of pl80^NACK can be made, wherein the resulting mutant retains at least one biological activity of pl80^NACK.

As the skilled artisan can appreciate, limited modifications can be made without destroying the biological function of a pl80^NACK polypeptide and only a portion of the entire primary sequence can be required for activity. For example, genetically engineered fragments of pl80^NACK either alone or fused to heterologous proteins such as fragments or fusion proteins that retain measurable activity in binding Notch or a Notch homologue, modulate the Notch- mediated target gene transcription, or other inherent biological activity of pl80^NACK fall within the definition of the polypeptide claimed as such. Moreover it is appreciated that minor modifications of the primary amino acid sequence of pl80^NΛCK can result in polypeptides which have substantially equivalent or enhanced function as compared to the murine pl80^NACK sequence set forth in SEQ ID NO : 2. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental such as through mutation in hosts harboring a pl80^NACK encoding nucleic acid. All such modified polypeptides are included in the definition of a pl80^NACK polypeptide as long as at least one biological function of pl80^NACK is retained. Further, various molecules can be attached to a pl80^NACK polypeptide including, for example, other polypeptides, carbohydrates, lipids, or chemical moieties. Such modifications are included within the definition of a pl80^NACK polypeptide.

The present invention also provides fragments of a pl80^NACK polypeptide. As used herein, the term fragment means a polypeptide fragment having substantially the same amino acid sequence as a portion of a pl80^NΛCK polypeptide. A fragment of the instant invention includes both biologically active fragments of pl80^NACK, e.g., fragments that bind to Notch and modulate Notch-mediated transcription, as well as immunogenic fragments of pl80^NACK for producing anti-pl80^NACK antibodies. In some embodiments, a fragment of pl80^NACK is defined as a peptide of 5 to 250 amino acid residues in length. In other embodiments, a fragment of pl80^NACK is defined as a peptide of 6 to 100 amino acid residues, 7 to 50 amino acid residues, or 10 to 25 amino acid residues in length. Particular embodiments of the present invention explicitly exclude from the definition of a fragment a polypeptide set forth in SEQ ID NO: 7. A pl80^NACK polypeptide, or fragment thereof, can be assayed for activity using one of the assays described herein or using another assay for measuring cell differentiation or cell survival known in the art. For example, a soluble pl80^NACK polypeptide or fragment thereof can be assayed by introducing an expression vector containing a nucleic acid molecule encoding the soluble pl80^NACK polypeptide or fragment into a cell and subsequently assaying for cell survival .

If desired, a pool of modified pl80^NACK polypeptide or pl80^NACK fragments can be assayed for activity en masse. For example, to identify an active fragment of pl80^NACK, pools of synthetic pl80^NACK fragments or pools of cell supernatants can be assayed for the ability to bind Notch; subsequently, pools of fragments or supernatants with activity can be subdivided, and the assay repeated in order to isolate the active modified pl80^NΛCK polypeptide or fragment from the active pool . An isolated pl80^NACK polypeptide, or fragment thereof, can be obtained by a variety of methods known within the art, including biochemical, recombinant and chemical synthesis methods. Biochemical methods for isolating a pl80^NACK polypeptide, or fragment thereof, include preparative gel electrophoresis, gel filtration, affinity chromatography, ion exchange and reversed phase chromatography, chromatofocusing, isoelectric focusing and sucrose or glycerol density gradients (see, for example, Chapter 38 of Deutscher, Methods in Enzymology: Guide to Protein Purification, Vol. 182, Academic Press, Inc., San

Diego (1990) and Chapter 8 of Balch et al . , Methods in

Enzymology, Vol. 257, Academic Press, Inc., San Diego

(1995)) . For example, as disclosed herein in Example 1, pl80^NACK was isolated by immunoprecipitation. Preparative gel electrophoresis can be useful in preparing an isolated pl80^NACK polypeptide or fragment of the invention. For example, a pl80^NACK polypeptide, or fragment thereof, can be isolated by preparative polyacrylamide gel electrophoresis and elution of the polypeptide or fragment by diffusion or electroelution (see, for example, Chapter 33 of Deutscher, supra, 1990) . Continuous elution gel electrophoresis using a system such as the Model 491 Prep Cell (BIORAD, Hercules, CA) can be used to purify a pl80^NACK polypeptide, or fragment thereof. If desired, continuous elution gel electrophoresis can be combined with further purification steps such as liquid phase preparative isoelectric focusing using, for example, the ROTOFOR system (BIO-RAD) . Affinity chromatography is particularly useful in preparing an isolated pl80^NACK polypeptide or fragment of the invention. A polypeptide that interacts with a pl80^NACK polypeptide, for example, a Notch polypeptide, can be useful as an affinity matrix for isolating a pl80^NACK polypeptide or active fragment of the invention. One skilled in the art understands that polypeptide fragments such as fragments of Notch also can be useful affinity matrices for isolating a pl80^NACK polypeptide or fragment of the invention.

Immunoaffinity chromatography can be particularly useful in isolating a pl80^NACK polypeptide or active fragment thereof. For example, immunoprecipitation or column chromatography with an antibody that selectively binds pl80^NACK can be used to isolate a pl80^NACK polypeptide or fragment thereof. An anti-pl80^NACK monoclonal or polyclonal antibody that selectively binds pl80^NACK can be prepared using an immunogen such as the sequence shown as SEQ ID NO : 2 , or a synthetic peptide fragment thereof, as described herein. One skilled in the art understands that a particularly useful immunogen can be a synthetic peptide fragment of SEQ ID NO : 2 having a sequence that is relatively unique to pl80^NACK. Thus, in selecting an immunogen, one can exclude, if desired, regions of SEQ ID NO : 2 which are conserved among other proteins. Methods of affinity chromatography are well-known in the art and are described, for example, in Chapters 29, 30 and 38 of Deutscher (1990) supra.

As exemplified herein, murine pl80^NACK was isolated affinity purification. T-cell lymphomas expressing a FLAG- tagged Nic were generated in mice by retroviral transduction mouse bone marrow cells. Following formation of lymphoma in mice (approximately 12 weeks) the lymphomatous tissue was harvested and prepared for purification of Notch complexes. Nic-associated proteins were purified by anti-FLAG affinity purification. To determine the identity of the Nic-associated proteins, the FLAG affinity eluate was separated on a polyacrylamide gel, bands were stained with colloidal blue, and individual polypeptides were excised from the gel and subjected to mass spectrometric analysis using LC MS/MS. From this analysis a protein with an apparent molecular weight of 180 kDa was identified, that specifically co-purified with Notch as well as with Maml2 and Maml3. A search of the database revealed the identity of the protein as "hypothetical" protein FLJ00269, i.e., an open reading frame predicted by genome sequence analysis. A pl80^NACK polypeptide or pl80^NACK fragment of the invention can also be produced by chemical synthesis, for example, by the solid phase peptide synthesis method of Merrifield, et al . (1964) J. Am. Chem. Soc. 85:2149. Standard solution methods well-known in the art also can be used to synthesize a polypeptide or fragment useful in the invention (see, e.g., Bodanszky (1984) Principles of Peptide Synthesis, Springer-Verlag, Berlin and Bodanszky (1993) Peptide Chemistry, Springer-Verlag, Berlin) . A newly synthesized polypeptide or fragment can be purified, for example, by high performance liquid chromatography (HPLC) and can be characterized using mass spectrometry or amino acid sequence analysis.

Recombinant methods for producing a polypeptide through expression of a nucleic acid sequence in a suitable host cell are well-known and routinely practiced in the art. Such methods are described, for example, in Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2nd Ed, VoIs 1 to 3, Cold Spring Harbor Laboratory Press, New York (1989) . Suitable expression vectors for use in eukaryotic and prokaryotic host cells can be readily obtained from commercial sources such as CLONTECH and STRATAGENE . By way of illustration, production of recombinant pl80^NACK polypeptide is provided in Example 2.

A recombinant pl80^NACK polypeptide or fragment of the invention can be expressed as a fusion protein with a heterologous "tag" for convenient isolation from bacterial or mammalian host proteins. For example, histidine-tagged recombinant pl80^NACK can be isolated by nickel-chelate chromatography. Similarly, a glutathione-S-transferase tag or an antigenic tag such as "FLAG, " "AU" or a myc epitope tag also can be included in a recombinant pl80^NACK polypeptide or fragment of the invention (Sambrook, et al . (1989) supra) .

A pl80^NACK nucleic acid molecule of the present invention includes the murine pl80^NACK cDNA set forth in SEQ ID NO : 1 , a nucleic acid molecule encoding a murine pl80^NACK polypeptide {e.g., having the sequence of SEQ ID N0:2) or a nucleic acid molecule encoding an ortholog of murine pl80^NACK polypeptide {e.g., having the sequence of SEQ ID NO : 3 , SEQ ID N0:4, SEQ ID NO : 5 , SEQ ID NO : 6 , or SEQ ID NO: 8) . The invention provides pl80^NACK nucleic acid molecules of at least 8 nucleotides in length (i.e., a hybridizable portion), as well as pl80^NACK nucleic acid molecules of 25, 50, 100, 150, or 200 (continuous) nucleotides in length, or a full-length pl80^NACK coding sequence. The invention also relates to nucleic acid molecules hybridizable to or complementary to the foregoing sequences or their complements. In some embodiments, nucleic acids are provided which encompass a sequence complementary to at least 10, 25, 50, 100, or 200 nucleotides or the entire coding region of a pl80^NACK. In a specific embodiment, a nucleic acid which is hybridizable to a mammalian pl80^NACK nucleic acid molecule {e.g., having sequence SEQ ID N0:l, or a 10, 25, 50, 100, or 200 nucleotide portion thereof), or to a nucleic acid encoding a pl80^NACK ortholog, under conditions of low stringency is provided. By way of example and not limitation, procedures using such conditions of low stringency are as follows (see also Shilo and Weinberg

(1981) Proc. Natl. Acad. Sci . USA 78:6789-6792): filters containing DNA are pretreated for 6 hours at 4O⁰C in a solution containing 35% formamide, 5X SSC, 50 mM Tris-HCl

(pH 7.5), 5 mM EDTA, 0.1% PVP, 0.1% FICOLL, 1% BSA, and 500 μg/ml denatured salmon sperm DNA. Hybridizations are carried out in the same solution with the following modifications: 0.02% PVP, 0.02% FICOLL, 0.2% BSA, 100 μg/ml salmon sperm DNA, 10% (wt/vol) dextran sulfate, and 5-20 x 10^G cpm ³²P-labeled probe is used. Filters are incubated in hybridization mixture for 18-20 hours at 40⁰C, and then washed for 1.5 hours at 55°C in a solution containing 2X SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The wash solution is replaced with fresh solution and incubated an additional 1.5 hours at 60⁰C. Filters are blotted dry and exposed for autoradiography. If necessary, filters are washed for a third time at 65-68⁰C and re-exposed to film. Other conditions of low stringency which may be used are well-known in the art (e.g., as employed for cross-species hybridizations) .

In another embodiment, a nucleic acid which is hybridizable to a mammalian pl80^NACK nucleic acid molecule under conditions of high stringency is provided. By way of example and not limitation, procedures using such conditions of high stringency are as follows: prehybridization of filters containing DNA is carried out for 8 hours to overnight at 65⁰C in buffer composed of 6X SSC, 50 πiM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02% FICOLL, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA. Filters are hybridized for 48 hours at 65⁰C in prehybridization mixture containing 100 μg/ml denatured salmon sperm DNA and 5-20 x 10^s cpm of ³²P-labeled probe. Washing of filters is done at 37°C for 1 hour in a solution containing 2X SSC, 0.01% PVP, 0.01% FICOLL, and 0.01% BSA. This is followed by a wash in 0. IX SSC at 5O⁰C for 45 minutes before autoradiography. Other conditions of high stringency which can be used are well known in the art.

Nucleic acids encoding fragments of pl80^NACK and modified pl80^NACK polypeptides, antisense, and pl80^NΛCK iRNA nucleic acids are additionally provided. As used herein, a nucleic acid molecule encoding a fragment or portion of a pl80^NACK polypeptide shall be construed as referring to a nucleic acid encoding only the recited fragment or portion of the pl80^NACK polypeptide and not the other contiguous portions of the pl80^NACK polypeptide.

Nucleic acid molecules such as antisense and iRNA (e.g., siRNA and shRNA) which hybridize or interfere with the expression pl80^NACK polypeptide are also embraced by the present invention. Synthetic siRNA, or pairs of short complimentary RNA molecules, or a single inverted small hairpin RNA (shRNA) are useful for RNAi -mediated inhibition of expression. The identification of suitable iRNA target regions in a nucleic acid of interest is routinely practiced in the art using commercially available algorithms (see, e.g., Khvorova, et al . (2003) Cell 115 (2) :209-16; Schwarz, et al (2003) Cell 115 (2) : 199-208 ; Reynolds, et al . (2004) Nat. Biotechnol . 22 (3 ): 326-30) . For example, kits for production of dsRNA for use in RNAi are available commercially, e.g., from New England Biolabs, Inc. and AMBION Inc. (Austin, TX). Methods of transfection of dsRNA or plasmids engineered to make dsRNA are routine in the art. When employing an siRNA molecule, said molecules are generally 19-30 nucleotides in length and have a GC content of 40-60%. Examples of siRNA molecules useful in decreasing the expression of a pl80^NACK polypeptide are provided in Table 2.

TABLE 2

It is expected the use of one or more of the molecules listed in Table 2 will provide a 40%, 50%, 60%, 70%, or higher reduction in the level of pl80^NACK polypeptide in a cell.

Silencing effects similar to those produced by RNAi have been reported in mammalian cells with transfection of an mRNA-cDNA hybrid construct (Lin, et al . (2001) Biochem. Biophys . Res. Commun. 281:639-44), providing yet another strategy for silencing a coding sequence of interest.

Cloning of a pl80^NACK nucleic acid molecule of the present invention can be carried out using methods routinely practiced in the art, including but not limited to expression cloning, nucleic acid hybridization and PCR amplification. For expression cloning, an expression library is constructed by isolating mRNA (e.g., human), producing cDNA and ligating the cDNA into an expression vector (e.g., a bacteriophage derivative) such that it is capable of being expressed by the host cell into which it is then introduced. Various screening assays can then be used to select for the expressed pl80^NACK product. In one embodiment, anti-pl80^NACK antibodies can be used for selection.

In another embodiment, PCR is used to amplify a pl80^NACK nucleic acid molecule. Oligonucleotide primers representing known pl80^NACK sequences (desirably mammalian sequences) are used as primers in PCR. In one embodiment, the oligonucleotide primers represent at least a part of the nucleic acid molecule encoding a pl80^NACK conserved motif of substantial amino acid sequence identity with murine pl80^NACK. Synthetic oligonucleotides can be utilized as primers to PCR amplify a pl80^NACK nucleic acid molecule using an RNA or DNA, e.g., a cDNA or genomic library, from any source as template. PCR can be carried out, e.g., by use of a commercial thermal cycler and Taq polymerase. One can choose to synthesize several different degenerate primers, for use in the PCR reactions. It is also possible to vary the stringency of hybridization conditions used in priming the PCR reactions, to allow for greater or lesser degrees of nucleic acid sequence similarity between the known P₁QQ^NACK _nuc]__e-j__{c aC}χd sequence and the nucleic acid ortholog being isolated. For cross-species hybridization, low stringency conditions are desirable. For same species hybridization, moderately stringent conditions are useful. After successful amplification of a segment of a pl80^NACK ortholog, that segment can be cloned and sequenced, and utilized as a probe to isolate a complete cDNA or genomic clone. This, in turn, permits the determination of the gene's complete nucleic acid sequence, the analysis of its expression, and the production of its protein product for functional analysis. In this fashion, additional nucleic acid molecules encoding pl80^NACK proteins are identified.

Any cell potentially can serve as the nucleic acid source for the molecular cloning of a pl80^NACK nucleic acid molecule of the present invention. The nucleic acid molecule encoding pl80^NACK can be isolated from mammalian, human, porcine, bovine, feline, avian, equine, canine, and rat, as well as additional primate sources, etc. The DNA from these source can be obtained by standard procedures known in the art for cloning DNA {e.g., a DNA library), by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell. (See, for example, Sambrook et al . (1989) Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.; Glover

(ed.) (1985) DNA Cloning: A Practical Approach, MRL Press,

Ltd., Oxford, U.K. VoIs. I and II). Clones derived from genomic DNA can contain regulatory and intron DNA regions in addition to coding regions. Whatever the source, the nucleic acid molecule is generally cloned into a suitable vector for propagation of the nucleic acid molecule.

In the molecular cloning of a nucleic acid molecule from genomic DNA, DNA fragments are generated, some of which will encode the desired gene. The DNA can be cleaved at specific sites using various restriction enzymes. Alternatively, one can use DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication. The linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography. Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired gene can be accomplished in a number of ways. For example, if an amount of a portion of a pl80^NACK (of any species) nucleic acid molecule or its specific RNA, or a fragment thereof, is available and can be purified and labeled, the generated DNA fragments can be screened by nucleic acid hybridization to the labeled probe (Benton and Davis (1977) Science 196:180; Grunstein and Hogness (1975) Proc. Natl. Acad. Sci . USA 72:3961). Those DNA fragments with substantial homology to the probe will hybridize. It is also possible to identify the appropriate fragment by restriction enzyme digestion (s) and comparison of fragment sizes with those expected according to a known restriction map if such is available. Further selection can be carried out on the basis of the properties of the gene. Alternatively, the presence of the gene can be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, cDNA clones, or DNA clones which hybrid-select the proper mRNAs, can be selected which produce a protein that, e.g., has similar or identical electrophoretic migration, isolectric focusing behavior, proteolytic digestion maps, binding activity, in vitro aggregation activity or antigenic properties as known for pl80^NACK. The pl80^NACK polypeptide can also be identified by binding of labeled antibody to the putatively pl80^NACK synthesizing clones, in an ELISA-type procedure .

A pl80^NACK nucleic acid molecule can also be identified by mRNA selection by nucleic acid hybridization followed by in vitro translation. In this procedure, fragments are used to isolate complementary mRNAs by hybridization. Such DNA fragments may represent available, purified pl80^NACK DNA of another species {e.g., human). Immunoprecipitation analysis or functional assays (e.g., binding to Notch; see infra) of the in vitro translation products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments that contain the desired sequences. In addition, specific mRNAs can be selected by adsorption of polysomes isolated from cells to immobilized antibodies specifically directed against pl80^NACK polypeptide. A radiolabelled pl80^NACK cDNA can be synthesized using the selected mRNA

(from the adsorbed polysomes) as a template. The radiolabelled mRNA or cDNA can then be used as a probe to identify the pl80^NACK nucleic acid fragments from among other genomic DNA fragments.

Alternatives to isolating the pl80^NACK genomic DNA include, but are not limited to, chemically synthesizing the gene sequence itself from a known sequence or making cDNA to the mRNA which encodes the pl80^NACK polypeptide. For example, RNA for cDNA cloning of the pl80^NACK nucleic acid molecule can be isolated from cells which express pl80^NACK. Other methods are possible and within the scope of the invention. A nucleic acid molecule of the present invention can also be mutated to encode for a modified pl80^NACK polypeptide or fragment, for example, by site-directed mutagenesis (see Wu (Ed.) Meth. Enzymol . Vol. 217, San Diego: Academic Press (1993) or Chapter 22 of Innis et al . (Ed.), PCR Protocols, San Diego: Academic Press, Inc. (1990) . Such mutagenesis can be used to introduce a specific, desired amino acid substitution, deletion or insertion; alternatively, a nucleic acid sequence can be synthesized having random nucleotides at one or more predetermined positions to generate random amino acid substitutions. Scanning mutagenesis also can be useful in generating nucleic acid molecules encoding pl80^NACK polypeptides or fragments that are modified throughout the entire polypeptide or fragment sequence. Such modified fragments can be screened for the ability to bind Notch or modify Notch mediated transcription.

A pl80^NACK polypeptide of the invention is useful for preparing an antibody that selectively binds a pl80^NACK polypeptide such as murine pl80^NACK (8EQ ID NO: 2) and/or a _pl QQ^NACK _orthoi_og (_SEQ _ID NO: 3, SEQ ID NO _: 4 , SEQ ID NO _: 5 , SEQ ID N0:6, SEQ ID NO:8). An antibody that selectively binds a pl80^NACK polypeptide can be useful, for example, in purifying a pl80^NACK polypeptide by immunoaffinity chromatography. Such an antibody also can be useful in screening assays for identifying small molecule inhibitors which compete for binding of the antibody to pl80^NACK polypeptide.

As used herein, the term antibody is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as polypeptide fragments of antibodies that retain selective binding activity for a pl80^NACK polypeptide of at least about 1 x 10⁵ M^"1. As the skilled artisan can appreciated, anti-pl80^NACK antibody fragments such as Fab, F(ab')₂ and Fv fragments can retain selective binding activity for a pl80^NACK polypeptide and, thus, are included within the definition of an antibody. In addition, the term antibody as used herein includes naturally occurring antibodies as well as non-naturally occurring antibodies and fragments that have binding activity such as chimeric antibodies or humanized antibodies. Such non- naturally occurring antibodies can be constructed using solid phase peptide synthesis or produced recombinantly . Such non-naturally occurring antibodies also can be obtained, for example, by screening combinatorial libraries composed of variable heavy chains and variable light chains as described by Borrebaeck (Ed.) (1995) Antibody Engineering (Second edition) New York: Oxford University Press .

An antibody selective for a polypeptide, or that selectively binds a polypeptide, binds with substantially higher affinity to that polypeptide than to an unrelated polypeptide. An antibody selective for a polypeptide also can be selective for a related polypeptide (e.g., an ortholog) . For example, an antibody selective for murine p₁₈₀ ^NAcκ (_SEQ _{ID N0:}2) also can be selective for human pl80^NACK (SEQ ID NO: 3) or for pl80^NACK orthologs from other species {e.g., as listed in Table 1) .

An anti-pl80^NACK antibody can be prepared, for example, using a pl80^NACK fusion protein or a synthetic peptide encoding a portion of pl80^NACK polypeptide such as murine

(SEQ ID NO: 2) as an immunogen. As indicated herein, a purified pl80^NACK polypeptide can be prepared from natural sources or produced recombinantly as described above, or fragments of pl80^NACK polypeptide, including a peptide portion of pl80^NACK such as a synthetic peptide, can be used as an immunogen. Examples of immunogenic fragments of murine and human pl80^NACK polypeptide are set forth in Table 3. TABLE 3

Protein cleavage prediction was carried out using FRAGPREDICT and MHC binding matrices using SYFPEITHI.

Non- immunogenic fragments or synthetic peptides of pl80^NACK can also be made immunogenic by coupling the hapten to a carrier molecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) . In addition, various other carrier molecules and methods for coupling a hapten to a carrier molecule are well-known in the art and described, for example, by Harlow and Lane (1988) Antibodies : A Laboratory Manual (Cold Spring Harbor Laboratory Press) .

The present invention also embraces vectors and host cells containing a recombinant nucleic acid molecule having a nucleotide sequence encoding a pl80^NACK polypeptide, or fragment thereof. The cell can be a prokaryotic cell or a eukaryotic cell such as an HEK293 cell, H1299 cell, COS cell or BHK cell. Such vectors and cells are useful for expressing a recombinant pl80^NACK polypeptide, or fragment thereof, for purification and production of antibodies as well as for determining the crystal structure of the catalytic domain.

A host cell transformed with a nucleic acid molecule encoding a pl80^NACK polypeptide can also be used in in vivo screening assay for determining whether a test agent modulates Notch-mediated signal transduction. As used herein, Notch-mediated signal transduction relates to the direct interaction between Notch and a DSL ligand which results in proteolytic processing that releases N^1C from the plasma membrane, wherein N^1C translocates to the nucleus and interacts with the DNA binding protein CSL and the co- activator protein Mastermind- likel (Mamll) to activate transcription of target genes. As exemplified herein, Notch-mediated signal transduction can be determined by reporter gene expression (e.g., a CSL-responsive promoter fused to luciferase) the presence of a test agent and absence of a test agent (i.e., control) . Agents identified in accordance with this in vivo assay can include agents which directly interact with the pl80^NACK polypeptide or modulate the expression of nucleic acid molecules encoding pl80^NACK polypeptide thereby changing the amount of the protein in the cell and therefore Notch-mediated signal transduction. Alternatively in vitro screening assays can be carried out which are based upon kinase activity or binding between Notch, or other component of a Notch complex (e.g., Mamll or CSL), and pl80^NACK polypeptide. Such assays generally involve contacting Notch, or a protein of the Notch complex, with pl80^NACK polypeptide in the presence or absence of a test agent and determining whether the kinase activity of pl80^NACK polypeptide is modulated or whether the interaction between Notch, or a protein of the Notch complex, with pl80^NΛCK polypeptide is disrupted {e.g., via co-immunoprecipitation assays) .

The in vitro and in vivo screening assays disclosed herein can be performed in any format that allows rapid preparation and processing of multiple reactions such as in, for example, multi-well plates of the 96-well variety. Stock solutions of test agents as well as assay components are prepared manually and all subsequent pipetting, diluting, mixing, washing, incubating, sample readout and data collecting is done using commercially available robotic pipetting equipment, automated work stations, and analytical instruments for detecting the signal generated by the assay.

In addition to pl80^NACK polypeptide, Notch, or a protein of the Notch complex, a variety of other reagents can be included in the screening assays. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc. which can be used to facilitate optimal protein- protein binding and/or reduce non-specific or background interactions. Also, reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti -microbial agents, and the like can be used. The mixture of components can be added in any order that provides for the requisite binding. When assaying test agents in the binding and cell- based assays of the instant invention, it is desirable that a control be included for comparison. For example, a control can be a test reaction which lacks the test agent or a control reaction can be test reaction which contains a known agent which has a high affinity for binding and inhibiting Notch-mediated signal transduction.

Test agents which can be screened in accordance with the methods of the present invention are generally derived from libraries of agents or compounds. Such libraries can contain either collections of pure agents or collections of agent mixtures. Examples of pure agents include, but are not limited to, proteins, polypeptides, peptides, antibodies, nucleic acids, iRNA, antisense oligonucleotides, carbohydrates, lipids, synthetic or semisynthetic chemicals, and purified natural products. Examples of agent mixtures include, but are not limited to, extracts of prokaryotic or eukaryotic cells and tissues, as well as fermentation broths and cell or tissue culture supernates. In the case of agent mixtures, the methods of this invention are not only used to identify those crude mixtures that possess the desired activity, but also provide the means to monitor purification of the active agent from the mixture for characterization and development as a therapeutic drug. In particular, the mixture so identified can be sequentially fractionated by methods commonly known to those skilled in the art which can include, but are not limited to, precipitation, centrifugation, filtration, ultrafiltration, selective digestion, extraction, chromatography, electrophoresis or complex formation. Each resulting subfraction can be assayed for the desired activity using the original assay until a pure, biologically active agent is obtained. Additional screens such as well-established computational screens are also contemplated for use in conjunction with the screening method disclosed herein.

Agents identified in accordance with the assay method of the present invention will be useful in various applications including inhibiting Notch-mediated signaling as well as anti-neoplastic therapeutics with specificity to tumors harboring mutations in the Notch pathway. Examples of tumors exhibiting aberrant Notch activity include, but are not limited to, lymphoma, melanoma, and cancers of the breast, lung, colon, central nervous system and pancreas. Furthermore, manipulation of Notch activity through pl80^NACK protein is useful in regulating stem cell growth and differentiation. To evaluate the efficacy of an agent identified using the screening method of the invention, one of skill will appreciate that a model system of any particular disease or disorder involving Notch-mediated signal transduction can be utilized to evaluate the adsorption, distribution, metabolism and excretion of a compound as well as its potential toxicity in acute, sub-chronic and chronic studies .

The use of an agent identified in accordance with the assay method of the present invention in the prevention or treatment of a disease or condition involving Notch- mediated signal transduction typically involves the steps of first identifying a patient at risk of having or having a disease or disorder involving Notch-mediated signal transduction {e.g., lymphoma, melanoma, and cancers of the breast, lung, colon, central nervous system and pancreas) . Once such an individual is identified using, for example, standard clinical practices, said individual is administered a pharmaceutical composition containing an effective amount of an agent identified in the screening methods of the invention. In most cases this will be a human being, but treatment of agricultural animals, e.g., livestock and poultry, and companion animals, e.g., dogs, cats and horses, is expressly covered herein. The selection of the dosage or effective amount of an agent is that which has the desired outcome of reducing at least one sign or symptom of a disease or disorder involving Notch-mediated signal transduction in a patient. Pharmaceutical compositions can be in the form of pharmaceutically acceptable salts and complexes and can be provided in a pharmaceutically acceptable carrier and at an appropriate dose. Such pharmaceutical compositions can be prepared by methods and contain carriers which are well-known in the art. A generally recognized compendium of such methods and ingredients is Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro, editor, 20th ed. Lippincott Williams & Wilkins: Philadelphia, PA, 2000. A pharmaceutically-acceptable carrier, composition or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, is involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.

Examples of materials which can serve as pharmaceutically acceptable carriers include sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffered solutions; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

Pharmaceutical compositions appropriately formulated for parenteral (for example, by intravenous, intraperitoneal, subcutaneous or intramuscular injection), topical (including buccal and sublingual), oral, intranasal, intravaginal , or rectal administration can be prepared according to standard methods.

The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular agent being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular agent employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of an agent at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Moreover, given the efficacy of an VEGFRl siRNA developed by Sirna Therapeutics (San Francisco, CA) for the treatment of AMD, one of skill in the art can appreciate dosing of siRNAs useful for achieving the desired therapeutic result with no systemic or local adverse events.

The invention is described in greater detail by the following non- limiting examples.

Example 1: Purification of pl80^NACK Polypeptide

Nuclear extracts of FLAG-fusion-N^lc tumors (50 mg) were incubated with 100 μl of anti-FLAG M2 affinity gel (SIGMA, St. Louis, MO) overnight at 4⁰C. Beads were washed with 3 x 5 ml BC500 buffer (20 mM Tris, pH 8; 0.5 M KCl; 10% glycerol; 1 mM EDTA; 1 mM DTT; 0.1% NP-40; 0.5 mM PMSF; and aprotinin, leupeptide, and pepstatin at 1 μg/ml each) and 2 times with 5 ml BClOO buffer (20 mM Tris, pH 8; 0.1 M KCl, 10% glycerol; 1 mM EDTA; 1 mM DTT; 0.1% NP-40; and aprotinin, leupeptide, and pepstatin at 1 μg/ml each) . Bound peptides were eluted with 500 μg/ml FLAG peptide (SIGMA) in BClOO buffer.

Example 2: Cloning of Nucleic Acids Encoding pl80^NACK Using PCR, mouse and human cDNAs encoding FLJ00269 protein were amplified and cloned into commercially available expression vectors. Sequence and expression were validated. From the sequence analysis, it was determined that the full 5' region of FLJ00269 encoded an additional 195 amino acids not predicted by the genome project, likely due to sequencing errors. SEQ ID NO : 1 represents the complete coding sequence and amino acid sequence of mouse FLJ00269, designated herein as pl80^NACK.

Example 3: Co-Immunoprecipitation

Since pl80^NACK was purified with Notch from a complex sample, the association between these two proteins was subsequently evaluated. To demonstrate an in vivo interaction between pl80^NACK and N^1C, co-immunoprecipitation experiments were performed. Expression vectors encoding pl80^NACK and Notch were co-transfected into HEK293 cells. Forty-eight hours after transfection, cells were lysed for 30 minutes at 4°C using NP-40 Lysis buffer (150 mM NaCl, 50 mM HEPES, pH 7.4, 1.5 mM EDTA, 10 % glycerol, 1 % NP-40, supplemented with 0.5 mM DTT, 0.2 mM PEFABLOC, 1 μg/ml leupeptin, 1 μg/ml aprotinin (ROCHE), 50 mM NaF and 0.5 mM Vanadate) . After centrifugation, supernatants were incubated overnight at 4°C with FLAG M2 beads. The immuno- complexes were washed extensively with lysis buffer, and the precipitates were boiled in Laemmli buffer and assayed by western blot analysis using an anti -Notch antibody. This analysis revealed the presence of a 100 kDa band corresponding to N^1C . As a control, neither pl80^NACK nor N^ic immunoprecipitated with IgG. This analysis confirmed that N^ic interacts with pl80^NACK.

Example 4: Luciferase Reporter Assay The observed co-immunoprecipitation between N^1C and pl80^NACK indicated that pl80^NACK was targeted specifically to CSL target genes to facilitate efficient transcriptional activation by Notch. Transcription activation by Notch is essential for its normal biological and oncogenic activities. Therefore it was determined whether pl80^NACK could modulate Notch-mediated-CSL target gene activity. To demonstrate this activity, an 8xCSL luciferase reporter that contains multiple copies of an optimal CSL-binding site was employed.

H1299 cells were seeded on six well plates at 100,000 cells per well one day before transfection and then transfected with various combinations of expression plasmid DNA corresponding to a final amount of 2 μg of DNA. The total amount of plasmid was maintained constant by adding appropriate amounts of empty vector without insert . The transfected cells were harvested at 48 hours post- transfection and luciferase activities were measured using a commercially available luciferase reporter assay system. Luciferase values were corrected for transcription efficiency by normalizing to β-galactosidase activity.

Transfection of pl80^NACK in H1299 cells increased CSL- directed luciferase activity 2-fold compared to N^1C alone and 6 -fold when Mastermind and pl80^NACK were added (Figure 2) . These results indicate that pl80^NACK acts as a specific co-activator for Notch transcription. Taken together, the results disclosed herein clearly indicate that pl80^NACK is a critical component of the Notch pathway and tumor cell survival. Furthermore pl80^NACK, by virtue of functionality with Notch, is a valid therapeutic target.

Claims

What is claimed is:

1. An isolated pl80^NACK polypeptide or fragment thereof.

2. The pl80^NACK polypeptide or fragment of claim 1, wherein said polypeptide or fragment is recombinantly produced.

3. An isolated nucleic acid molecule encoding the pl80^NACK polypeptide of claim 1.

4. An isolated nucleic acid molecule which hybridizes to the nucleic acid molecule of claim 3.

5. A vector comprising the nucleic acid molecule of claim 3.

6. A host cell comprising the vector of claim 5.

7. An antibody that selectively binds the pl80^NACK polypeptide or fragment of claim 1.

8. A fusion protein comprising a heterologous tag fused to the pl80^NACK polypeptide or fragment of claim 1.

9. A method for identifying an agent that modulates Notch-mediated signal transduction comprising contacting a pl80^NACK polypeptide or cell expressing said polypeptide with a test agent and determining whether said agent modulates the expression or activity of the pl80^WACK polypeptide thereby identifying an agent that modulates Notch-mediated signal transduction.

10. A method for modulating Notch-mediated signal transduction comprising contacting a cell with an agent identified by the method of claim 9 thereby modulating Notch-mediated signal transduction.