WO2007005635A2

WO2007005635A2 - Mitotic spindle protein aspm as a diagnostic marker for neoplasia and uses therefor

Info

Publication number: WO2007005635A2
Application number: PCT/US2006/025640
Authority: WO
Inventors: Paul K. Goldsmith; Vladimir Larionov; Natalay Kouprina; John I. Risinger
Original assignee: Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2005-07-01
Filing date: 2006-06-29
Publication date: 2007-01-11
Also published as: WO2007005635A3

Abstract

The invention features diagnostic and therapeutic methods and compositions featuring ASPM proteins and nucleic acid molecules whose expression is increased in neoplastic tissues.

Description

MITOTIC SPINDLE PROTEIN ASPM AS A DIAGNOSTIC MARKER FOR NEOPLASIA AND USES THEREFOR

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of the following U.S. Provisional Application No.:

60/696,212, which was filed on July 1, 2005, the contents of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Research supporting this application was carried out by the United States of America as represented by the Secretary, Department of Health and Human Services.

BACKGROUND OF THE INVENTION Nearly 23 % of deaths in the United States in 2001 were cancer related (American

Cancer Society, 2001 Statistics). Gynecological cancers, such as uterine carcinoma and ovarian cancer are critical problems for women. Uterine cancer is the most common cancer in the United States among women. Ovarian cancer ranks fifth in cancer deaths among women, totaling more deaths than any other cancer of the female reproductive system. This may reflect the lack of a diagnostic test capable of diagnosing ovarian cancer in its early stages when it is most amenable to treatment. The effective treatment of ovarian cancer will likely be significantly increased if a screening test to identify early stage ovarian cancer is developed. Given the high incidence of ovarian cancer and other neoplasias, there is an urgent need for new diagnostic and therapeutic methods.

SUMMARY OF THE INVENTION

In general, the present invention provides for diagnostic methods, compositions, and kits that are useful for identifying a neoplasia by measuring Abnormal SPindle-like Microcephaly associated (ASPM) expression in a patient sample. In addition, the invention provides for methods of treating a neoplasia having increased ASPM expression.

In one aspect, the invention generally features a method of diagnosing a subject (e.g., a human) as having, or having a propensity to develop, a neoplasia (e.g., cancer of the brain, breast, lung, pancreas, uterus, colon, thyroid, liver, bladder, kidney, ovary, testis, stomach, lymph node, cervix, and esophagus). The method involves determining the level of expression of an ASPM nucleic acid molecule (e.g., any nucleic acid sequence provided herein) in a subject sample, where an increased level of expression relative to a reference, indicates that the subject has or has a propensity to develop a neoplasia.

In another aspect, the invention features a method of diagnosing a subject as having, or having a propensity to develop, a neoplasia. The method involves determining the level of expression of an ASPM polypeptide (e.g., any amino acid sequence provided herein) in a subject sample, where an increased level (e.g., increased by at least 5%, 10%, 25%, 50%, 75%, 85%, 90% or 95%; or where the increase is by 5X, 10X, 2OX or 30X) of expression relative to the level of expression in a reference, indicates that the subject has or has a propensity to develop a neoplasia. In one embodiment, the method involves determining the level of expression of the ASPM polypeptide (e.g., using an immunological assay).

In another aspect, the invention features a method of diagnosing a subject as having, or having a propensity to develop, a neoplasia. The method involves determining the level of biological activity (e.g., mitotic spindle activity, calmodulin binding activity, cell proliferative active, maintenance of chromosomal stability) of an ASPM polypeptide in a subject sample, where an alteration in the level of biological activity relative to the biological activity in a reference, indicates that the subject has or has a propensity to develop a neoplasia.

In yet another aspect, the invention features a method of monitoring a subject diagnosed as having a neoplasia. The method involves determining the level of an ASPM polypeptide in a subject sample, where an alteration in the level of expression relative to the level of expression in a reference indicates the severity of neoplasia in the subject. In one embodiment, the subject is being treated for a neoplasia (e.g. breast, prostate, lung, testis, ovary, or uterine neoplasia). In one embodiment, the alteration is an increase or a decrease. In a related embodiment, the increase indicates an increased severity of neoplasia. In another embodiment, the reference is a control subject sample (e.g., a biological sample, such as a biological fluid or tissue sample). In one embodiment, the reference is a sample obtained at an earlier time point.

In various embodiments of the above aspects, the method is used to diagnose a subject as having neoplasia; is used to determine the treatment regimen for a subject having neoplasia; is used to monitor the condition of a subject being treated for neoplasia; or is used to determine the prognosis of a subject having neoplasia. In a related embodiment, a poor prognosis determines an aggressive treatment regimen for the subject.

In another aspect, the invention provides a method for identifying a subject as having or having a propensity to develop a neoplasia. The method involves detecting an alteration in the sequence of an ASPM nucleic acid molecule relative to the sequence or expression of a reference molecule. In one embodiment, the alteration is detected using a hybridization reaction or is detected by sequencing the nucleic acid molecule.

In another aspect, the invention provides an ASPM antibody that specifically binds to an ASPM protein or fragment thereof (e.g., a VTRK. or QSPE epitope of an ASPM polypeptide).

In yet another aspect, the invention features a polypeptide containing an isolated ASPM protein variant, or fragment thereof, having substantial identity to ASPM variant 1, 2, or 3, where the polypeptide has an ASPM biological activity (e.g., mitotic spindle activity, calmodulin binding activity, functions in cell proliferation, or functions in chromosomal stability). In one embodiment, the ASPM protein variant is at least 80%, 85%, 90%, or 95% identical to ASPM variant 1, 2, or 3. In another embodiment, the ASPM protein variant contains at least an IQ domain and is capable of binding calmodulin. In yet another embodiment, the polypeptide consists of an ASPM protein variant selected from the group consisting of ASPM variant 1, 2, and 3. In various embodiments, the polypeptide is a fusion protein; is linked to a detectable amino acid sequence; or is linked to an affinity tag.

In another aspect, the invention features an isolated ASPM nucleic acid molecule, where the nucleic acid molecule encodes a polypeptide of any of the previous aspects, a vector comprising such a nucleic acid molecule, and a host cell (e.g., in vitro or in vivo) comprising the vector or nucleic acid molecule.

In a related aspect, the invention features an isolated ASPM inhibitory nucleic acid molecule, where the inhibitory nucleic acid molecule specifically binds at least a fragment of a nucleic acid molecule encoding an ASPM protein; a vector containing a nucleic acid molecule encoding the ASPM inhibitory nucleic acid molecule; and a host cell (e.g., in vitro or in vivo) comprising the vector or nucleic acid molecule.

In various embodiments of the previous aspects, the vector is an expression vector where the nucleic acid molecule is positioned for expression. In other embodiments, the nucleic acid molecule is operably linked to a promoter (e.g., a promoter suitable for expression in a mammalian cell). In yet other embodiments of any of the above aspects, the host cell expresses an ASPM protein variant. In still other embodiments, the host cell is a mammalian cell, such as a human cell.

In a related aspect, the invention features a double-stranded RNA corresponding to at least a portion of an ASPM nucleic acid molecule that encodes an ASPM protein, where the double-stranded RNA is capable of altering protein expression level. In one embodiment, the

RNA is an siRNA.

In another related aspect, the invention features an antisense nucleic acid molecule, where the antisense nucleic acid molecule is complementary to at least six nucleotides of an ASPM nucleic acid molecule that encodes an ASPM protein, and where the antisense is capable of altering expression from the nucleic acid molecule to which it is complementary. In another aspect, the invention features a primer capable of binding to an ASPM nucleic acid molecule encoding an ASPM protein variant that is upregulated in a neoplastic tissue. In a related aspect, the invention features a collection of primers capable of binding to and amplifying an ASPM nucleic acid molecule, where at least one of the primers in the collection is the primer of the previous aspect. In one embodiment, the collection features at least one pair of primers capable of amplifying an ASPM protein (e.g., ASPM, ASPM variant

1, 2, or 3). In another embodiment, the primers are useful in diagnosing a neoplasia. In yet another aspect, the invention features a pharmaceutical composition containing an effective amount of an ASPM protein, variant, or fragment thereof, in a pharmaceutically acceptable excipient, where the fragment is capable of modulating (i.e., increasing or decreasing) cell proliferation or chromosome stability.

In yet another aspect, the invention features a pharmaceutical composition containing an effective amount of a nucleic acid molecule of any previous aspect in a pharmaceutically acceptable excipient, where the fragment is capable of modulating cell proliferation or chromosomal stability.

In yet another aspect, the invention features a pharmaceutical composition containing an effective amount of a vector containing a nucleic acid molecule encoding an ASPM protein of any previous aspect in a pharmaceutically acceptable excipient, where expression of the ASPM protein in a cell is capable of reducing, stabilizing, or inhibiting a neoplasia, decreasing cell proliferation or enhancing chromosomal stability. In one embodiment, the

ASPM nucleic acid molecule is positioned for expression in a mammalian cell.

In another aspect, the invention features an ASPM biomarker purified on a biochip. In yet another aspect, the invention features a microarray containing at least two nucleic acid molecules, or fragments thereof, fixed to a solid support, where at least one of the nucleic acid molecules is an ASPM nucleic acid molecule. In a related aspect, the invention features a microarray containing at least two polypeptides, or fragments thereof, bound to a solid support, where at least one of the polypeptides on the support is an ASPM polypeptide.

In yet another aspect, the invention features a diagnostic kit for the diagnosis of a neoplasia in a subject containing an ASPM nucleic acid molecule, or fragment thereof, and written instructions for use of the kit for detection of a neoplasia.

In yet another aspect, the invention features a diagnostic kit for the diagnosis of a neoplasia in a subject containing an antibody that specifically binds an ASPM polypeptide, or fragment thereof, and written instructions for use of the kit for detection of a neoplasia. In yet another aspect, the invention features a kit identifying a subject as having or having a propensity to develop a neoplasia, containing an adsorbent, where the adsorbent retains an ASPM biomarker, and written instructions for use of the kit for detection of a neoplasia.

In yet another aspect, the invention features a kit containing a first capture reagent that specifically binds an ASPM biomarker, and written instructions for use of the kit for detection of a neoplasia.

In yet another aspect, the invention features a method for detecting neoplasia in a subject sample, where the method involves (a) contacting a subject sample with a capture reagent affixed to a substrate; and (b) capturing an ASPM polypeptide or nucleic acid molecule with the capture reagent. In one embodiment, the subject sample comprises an ASPM protein or fragment thereof. In another embodiment, the ASPM protein is fractionated prior to contacting the capture reagent.

In yet another aspect, the invention features a method of altering the expression of an ASPM nucleic acid molecule in a cell, the method comprising contacting the cell with an effective amount of a compound capable of altering the expression of the ASPM nucleic acid molecule. In one embodiment, the compound is an ASPM antisense nucleic acid molecule, a small interfering RNA (siRNA), or a double stranded RNA (dsRNA) that inhibits the expression of an ASPM nucleic acid molecule.

In yet another aspect, the invention features a method of altering ASPM protein expression in a cell, the method comprising contacting the cell with a compound capable of altering the expression of an ASPM polypeptide.

In various embodiments of the above aspects, the cell is a human cell, a neoplastic cell, a cell is in vitro or a cell in vivo. In yet another aspect, the invention features a method of treating or preventing a neoplasia. The method involves administering to a subject (e.g., a human) in need thereof an effective amount of a pharmaceutical composition that alters expression of an ASPM polypeptide. In yet another aspect, the invention features a method of identifying a compound that inhibits a neoplasia. The method involves contacting a cell that expresses an ASPM nucleic acid molecule with a candidate compound, and comparing the level of expression of the nucleic acid molecule in the cell contacted by the candidate compound with the level of expression in a control cell not contacted by the candidate compound, where an alteration in expression of the ASPM nucleic acid molecule identifies the candidate compound as a compound that inhibits a neoplasia.

In a related aspect, the invention features a method of identifying a compound that inhibits a neoplasia. The method involves contacting a cell that expresses an ASPM polypeptide with a candidate compound, and comparing the level of expression of the polypeptide in the cell contacted by the candidate compound with the level of polypeptide expression in a control cell not contacted by the candidate compound, where an alteration in the expression of the ASPM polypeptide identifies the candidate compound as a compound that inhibits a neoplasia.

In another related aspect, the invention features a method of identifying a compound that inhibits a neoplasia. The method involves contacting a cell that expresses an ASPM polypeptide with a candidate compound, and comparing the biological activity of the polypeptide in the cell contacted by the candidate compound with the level of biological activity in a control cell not contacted by the candidate compound, where an alteration in the biological activity of the ASPM polypeptide identifies the candidate compound as a candidate compound that inhibits a neoplasia.

In another related aspect, the invention features method of identifying a candidate compound that inhibits a neoplasia, the method comprising a) contacting a cell containing an ASPM nucleic acid molecule present in an expression vector that includes a reporter construct; b) detecting the level of reporter gene expression in the cell contacted with the candidate compound with a control cell not contacted with the candidate compound, where an alteration in the level of the reporter gene expression identifies the candidate compound as a candidate compound that inhibits a neoplasia.

In various embodiments of the above aspects, the alteration in expression is an increase or a decrease in transcription. In other embodiments, the alteration in expression is an increase or a decrease in translation. In still other embodiments, the cell is in vitro or in vivo. In still other embodiments, the cell is a human cell, such as a neoplastic cell. In yet other embodiments, the alteration in expression is assayed using an immunological assay, an enzymatic assay, or a radioimmunoassay. In various embodiments of the above aspects, the cell is a human cell, a neoplastic cell, a cell is in vitro or a cell in vivo. In various embodiments of any of the above-aspects, ASPM protein or nucleic acid molecule expression is increased in a neoplastic cell by at least 2X, 5X, 10X, 2OX, 30X, 4OX, 5OX the level of expression in a corresponding control cell. In various embodiments of any of the above aspects, a method or composition that alters (e.g., increases or decreases) ASPM expression by at least 5%, 10%, 25%, 50%, 75% or 100% is useful in the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures IA and IB show mouse and human ASPM expression in a variety of tissues. Figure IA is a photomicrograph showing ASPM expression in an E14 whole mouse embryo as detected by hybridization to antisense probe. Tissues are denoted using the following identifiers: Th=thalamus; Ma=⁼mammilary region; H=heart; Li=liver; Lu=lung; KHkidney. Figure IB is a photograph of an agarose gel showing relative levels of human ASPM expression in embryonic tissues. The ASPM transcripts in various tissues were analyzed using RT-PCR. Expression analyses of the mRNA was performed using human multiple fetal tissue cDNAs. β-actin was used as an internal control.

Figures 2A and 2B are graphs showing expression of ASPM ia ovarian and uterine cancers. Figure 2A shows ASPM expression as determined using real-time PCR in samples of normal ovary (n=4) and ovarian cancers (n=48). Figure 2B shows ASPM expression as determined using real-time PCR in samples of normal endometrium (n=6), endometrioid endometrial cancer(n=15), mixed mesodermal tumors (MMT) of the uterus (n=15), and serous endometrial carcinomas (n=15).

Figures 3 A and 3B show RT-PCR expression of ASPM in various human and mouse tissues. Figure 3A shows expression in human normal and matching tumor tissues. Figure 4B shows expression in normal murine tissues. RT-PCR expression of actin is provided as a reference.

Figures 4A and 4B are photographs of an agarose gel containing alternatively spliced ASPM variants in fetal tissues (Figure 4A) and β-actin (Figure 4B) visualized using ethidium bromide. Two major ASPM transcripts with sizes of ~10.3 and 5.7 kb were identified in all tissues analyzed. Additional spliced variants with variable sizes are also seen, β-actin was used as the internal control.

Figures 5A and 5B show ASPM spliced forms in human and mouse. Figure 5A is a schematic diagram that shows the positions of major domains in the ASPM protein. The putative microtubule-binding domain is in gray, the calponin-homology domain in orange (positions 960-1056 and 1114-1174), IQ repeats in blue, and the terminal domain in black. Yellow color marks positions of newly identified 32 and 35 as long repeats in the N-terminus (see B). The middle part shows major spliced variants and comparison with the full-length ASPM protein. The bottom part shows parts of the human ASPM protein encoded by individual exons. Spliced variant 2 contains exon 26 followed by a part of intron 26 where the ORF is disrupted by stop codon. To better separate individual exons, the odd numbered exons are colored in black and even numbered ones in white. Figure 5B shows sequence alignments among mammalian species for two ASPM repeats identified herein (SEQ ID Nos: 1-16). The human ASPM protein contains two repeats in positions 316-347 and 366-400. Thee repeats are found in other mammals and one is also preserved in chicken. Identical amino acids are identified in black boxes. Residues shown in upper case font are amino acids that are invariant among the species in the alignment. Residues shown in lower case font are amino acids that are conserved, but that are not invariant. Gray shading identifies conservative substitutions. Figures 6A-6D show the structure and evolution of human ASPM IQ repeats (SEQ ID

Nos: 17-97) (Figures 6 A, 6B, and 6C) and mouse IQ repeats Figure 6D. Figure 6 A shows the organization of IQ repeats in the human ASPM protein. At the far left of Figure 6A, the number of IQ repeats and their positions in the full-length ASPM protein is shown. The length of the individual repeat is also indicated by amino acid residue numbers. The human alternatively spliced variants are highlighted in orange. Note that variants 2 and 3 contain large deletions that extend further toward the C-terminus of ASPM. The blue box displays the region deleted in mouse and rat. Color is used to mark positions that are variable in the analyzed primates (green monkey, rhesus monkey, orangutan, gorilla, chimpanzee, and human) and also changes specific to African hominoids. Based on the Gonnet PAM250 matrix, substitutions were divided into noncoservative (P < 0.5), and conservative (the rest). The IQ repeats 4-54 form an organized array of longer (-27 aa) and shorter (~23 aa) units. Alignment and conservation is shown separately for long (Figure 6B) and short repeats (Figure 6C) from the IQ4-54 region. For both alignments, particular features (i.e., aliphatic, polar, hydrophobic, positive, and small) of the most conserved amino acids positions are indicated in Figures 6B and 6C. Figure 6D shows the structure and evolution of mouse ASPM IQ repeats. The left column shows the number of IQ repeats and their positions in the full-length ASPM protein; the right column shows the length of the individual repeats. The grey positions mark sites that are different between mouse and rat proteins.

Figures 7A-7I are photomicrographs showing the cytological analysis of ASPM proteins in mitotic cells HT1080 cells. HT1080 cells grown on coverslips were analyzed by indirect immunofluorescent staining using antibodies against human ASPM (Figures 7B, 7E, and 7H). Chromosomal DNA was counterstained with DAPI (Figures 7A, 7D and 7G). Anti-ASPM staining shows characteristic spindle pole staining patterns during mitotic periods, prometaphase (VTRK), metaphase (QSPE) and anaphase (VTRK). Scale bar indicates 10 μm.

Figures 7 J, 7K, and 7L show a characterization of the anti-VTKR antibody. Figure 7A shows the analysis of protein extracts from a normal and a microcephalic individuals that were run on an SDS PAGE gel, transferred to a membrane, and immunoblotted with the anti- VTKR antibody. The Western blot in Figure 7 J shows the presence of two predicted ASPM proteins (a full-size 410 kDa protein and a 218 kDa isoform lacking an exon 18-encoded segment) in a protein extract derived from normal human cells (HT1080) (human lane). The predicted isoforms are 35 kDa shorter (i.e., 385 kDa and 183 kDa) in MM10458 cell protein extracts, which are derived from a microcephalic individual (patient lane). ASPM is truncated by a frameshift in exon 24 in MMl 0458. The anti-VTKR antibody also visualizes at least three additional bands in the HT 1080 extract; two of these are missing in the MMl 0458 extract. These bands may correspond to the predicted 164 kDa and 124 kDa ASPM isoforms. Figure 7K shows an immunoblot analysis of mouse ASPM proteins using the anti-VTKR antibody. The largest immunoreactive band in the mouse cell extract apparently corresponds to the predicted full-size 364 kDa ASPM protein that is 46 kDa shorter than human ASPM. The second major band apparently corresponds to the predicted 212 kDa mouse ASPM isoform lacking the exon 18-encoding segment that is approximately the same size as human ASPM isoform (218 kDa). Note that the small size bands visible in Figure 7J (i.e., < 150 kDa) were run off the gel to see the difference between the full-size human ASPM and mouse ASPM proteins. Figure 7L is a Western blot analysis showing the specificity of the affinity-purified anti-VTKR antibody. The antibody recognizes a recombinant MBP-ASPM fusion protein expressed in the pMAL-p2X expression vector. Protein extract (100 μg) from bacterial cells was analyzed by SDS-PAGE, followed by immunoblotting using either anti-VTKR antibody (lanes 1-4) or anti-MBP antibody (lanes 5- 8). Lanes 1 and 5 correspond to the vector without insert. The predicted 60 kDa band corresponding to the fusion protein was detected. Lanes 2, 3, 4, 6, 7, and 8 correspond to individual transformants.

Figures 8A-8J shows the sequences of the human ASPM polypeptide (Figure 8A), its variants 1 (Figure 8C), 2 (Figure 8E), and 3 (Figure 8G), and the nucleic acid molecules encoding each of these polypeptides (Figures 8B, 8D, 8F, 8H). Figures 81 and 8 J provide the nucleic acid and amino acid sequences of murine Aspm.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By "ASPM polypeptide" is meant a protein or protein variant, or fragment thereof, that is substantially identical to at least a portion of GenBank Accession No. NP__060606 (human ASPM) and that has an ASPM biological activity (e.g., calmodulin binding, function in mitosis, cell proliferation, regulation of symmetric cell division, or chromosomal stability). By "ASPM nucleic acid molecule" is meant a polynucleotide encoding an ASPM polypeptide or variant, or fragment thereof.

By "ASPM biological activity" is meant calmodulin binding activity, function during cell division, maintenance of chromosome stability, regulation of symmetric cell division, or any other function at a mitotic spindle, function in a neoplastic cell, or ASPM antibody binding.

By "alteration" is meant a change relative to a reference. For example, a change (increase or decrease) in the expression levels of a gene or polypeptide as detected by standard art known methods such as those described above. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50%, 75%, 85%, 95% or greater change in expression levels. "

By "biomarker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder. By "affinity tag" is meant any moiety used for the purification of a protein or nucleic acid molecule to which it is fixed.

By "aggressive treatment regiment" is meant a treatment regiment consistent with the treatment of a disease having an increased risk of lethality or a poor prognosis for recovery. Such treatments include chemotherapy, surgery, radiotherapy of increased dosage or toxicity relative to the treatment of a disease having a lower risk of lethality or a better prognosis.

The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, for example, hydroxyproline, gamma-carboxyglutamate, and O-phosphoserine, phosphothreonine.

By an "amino acid analog" is meant a compound that has the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group (e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium), but that contains some alteration not found in a naturally occurring amino acid (e.g., a modified side chain); the term " amino acid mimetic" refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. Amino acid analogs may have modified R groups (for example, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. In one embodiment, an amino acid analog is a D-amino acid, a β- amino acid, or an N-methyl amino acid.

Amino acids and analogs are well known in the art. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

By "antibody" is meant any immunoglobulin polypeptide, or fragment thereof, having immunogen binding ability.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

By "detectable amino acid sequence" or "detectable moiety" is meant a composition that when linked with the nucleic acid or protein molecule of interest renders the latter detectable, via any means, including spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

A "labeled nucleic acid or oligonucleotide probe" is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic bonds, van der Waals forces, electrostatic attractions, hydrophobic interactions, or hydrogen bonds, to a label such that the presence of the nucleic acid or probe may be detected by detecting the presence of the label bound to the nucleic acid or probe.

An "expression vector" is a nucleic acid construct, generated recombinantly or synthetically, bearing a series of specified nucleic acid elements that enable transcription of a particular gene in a host cell. Typically, gene expression is placed under the control of certain regulatory elements, including constitutive or inducible promoters, tissue-preferred regulatory elements, and enhancers.

By "an effective amount" is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a neoplastic disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount. By "fragment" is meant a portion (e.g., at least 10, 25, 50, 100, 125, 150, 200, 250,

300, 350, 400, or 500 amino acids or nucleic acids) of a protein or nucleic acid molecule that is substantially identical to a reference protein or nucleic acid and retains the biological activity of the reference. In some embodiments the portion retains at least 50%, 75%, or 80%, or more preferably 90%, 95%, or even 99% of the biological activity of the reference protein or nucleic acid described herein.

A "host cell" is any prokaryotic or eukaryotic cell that contains either a cloning vector or an expression vector. This term also includes those prokaryotic or eukaryotic cells that have been genetically engineered to contain the cloned gene(s) in the chromosome or genome of the host cell.

By "inhibitory nucleic acid" is meant a double-stranded RNA, siRNA (short interfering RNA), shRNA (short hairpin RNA), or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule.

The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. Various levels of purity may be applied as needed according to this invention in the different methodologies set forth herein; the customary purity standards known in the art may be used if no standard is otherwise specified.

By "isolated nucleic acid molecule" is meant a nucleic acid (e.g., a DNA, RNA, or analog thereof) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule which is transcribed from a DNA molecule, as well as a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

"Microarray" means a collection of nucleic acid molecules or polypeptides from one or more organisms arranged on a solid support (for example, a chip, plate, or bead).

By "neoplasia" is meant any disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancer is an example of a neoplasia. Examples of cancers include, without limitation, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblasts leukemia, acute promyelocyte leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Lymphoproliferative disorders are also considered to be proliferative diseases.

By "nucleic acid" is meant an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid, or analog thereof. This term includes oligomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages as well as oligomers having non-naturally occurring portions which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced stability in the presence of nucleases. Specific examples of some nucleic acids envisioned for this invention may contain phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Also preferred are oligonucleotides having morpholino backbone structures (Summerton, J. E. and Weller, D. D., U.S. Pat. No: 5,034,506). In other preferred embodiments, such as the protein- nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (P. E. Nielsen et al. Science 199: 254, 1997). Other preferred oligonucleotides may contain alkyl and halogen-substituted sugar moieties comprising one of the following at the 2' position: OH, SH, SCH3, F₅ OCN, O(CH2)_nNH2 or O(CH2)_n CH3, where n is from 1 to about 10; Ci to Cio lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O~, S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a conjugate; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group. Other preferred embodiments may include at least one modified base form. Some specific examples of such modified bases include 2-(amino)adenine, 2-(methylamino)adenine, 2- (imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine, or other heterosubstituted alkyladenines.

By "operably linked" is meant that a first polynucleotide is positioned adjacent to a second polynucleotide that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide. By "ortholog" is meant any protein or nucleic acid molecule of an organism that is highly related to a reference protein or nucleic acid sequence from another organism.

By "recombinant" is meant the product of genetic engineering or chemical synthesis. By "protein" is meant any chain of amino acids, or analogs thereof, regardless of length or post-translational modification.

By "positioned for expression" is meant that the polynucleotide of the invention (e.g., a DNA molecule) is positioned adjacent to a DNA sequence that directs transcription and translation of the sequence (i.e., facilitates the production of, for example, a recombinant protein of the invention, or an RNA molecule).

By "reference" is meant a standard or control condition.

By "siRNA" is meant a double stranded RNA. Optimally, an siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3' end. These dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream. Such siRNAs are used to downregulate mRNA levels or promoter activity.

By "specifically binds" is meant a molecule (e.g., peptide, polynucleotide) that recognizes and binds a protein or nucleic acid molecule of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a protein of the invention.

By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline. By "substantially identical" is meant a protein or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and most preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e'3 and e^~100 indicating a closely related sequence.

By "transformed cell" is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a polynucleotide molecule encoding (as used herein) a protein of the invention.

Methods of the Invention

The invention features compositions and methods useful for the diagnosis of a neoplasia in a subject. These methods and compositions are based, in part, on the discovery that ASPM polypeptide (e.g., ASPM, ASPM variant 1, 2, or 3) expression is increased in neoplastic tissues. In addition, the invention also provides methods and compositions for altering ASPM expression in a neoplastic cell. Such compositions and methods are likely to be useful for the treatment of neoplasia. Diagnostics

Neoplastic tissues express higher levels of an ASPM polypeptide (e.g., ASPM, ASPM variant 1, 2, or 3) or an ASPM polynucleotide than corresponding normal tissues. Accordingly, expression levels of an ASPM nucleic acid molecule or polypeptide are correlated with a particular disease state (e.g., neoplasia), and thus are useful in diagnosis. Accordingly, the present invention provides a number of diagnostic assays that are useful for the identification or characterization of a neoplasia.

In one embodiment, a patient having a neoplasia will show an increase in the expression of an ASPM nucleic acid molecule. Alterations in gene expression are detected using methods known to the skilled artisan and described herein. Such information can be used to diagnose a neoplasia. In another embodiment, an alteration in the expression of an ASPM nucleic acid molecule is detected using real-time quantitative PCR (Q-rt-PCR) to detect changes in gene expression.

Primers used for amplification of an ASPM nucleic acid molecule, including but not limited to those primer sequences described herein, are useful in diagnostic methods of the invention. The primers of the invention embrace oligonucleotides of sufficient length and appropriate sequence so as to provide specific initiation of polymerization on a significant number of nucleic acids. Specifically, the term "primer" as used herein refers to a sequence comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and most preferably more than 8, which sequence is capable of initiating synthesis of a primer extension product, which is substantially complementary to a locus strand. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent for polymerization. The exact length of primer will depend on many factors, including temperature, buffer, and nucleotide composition. The oligonucleotide primer typically contains between 12 and 27 or more nucleotides, although it may contain fewer nucleotides. Primers of the invention are designed to be "substantially" complementary to each strand of the genomic locus to be amplified and include the appropriate G or C nucleotides as discussed above. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions that allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with the 5' and 3' flanking sequences to hybridize therewith and permit amplification of the genomic locus. While exemplary primers are provided herein, it is understood that any primer that hybridizes with the target sequences of the invention are useful in the method of the invention for detecting ASPM nucleic acid molecules. In one embodiment, ASPM-specific primers amplify a desired genomic target using the polymerase chain reaction (PCR). The amplified product is then detected using standard methods known in the art. In one embodiment, a PCR product (i.e., amplicon) or real-time PCR product is detected by probe binding. In one embodiment, probe binding generates a fluorescent signal, for example, by coupling a fluorogenic dye molecule and a quencher moiety to the same or different oligonucleotide substrates (e.g., TaqMan® (Applied Biosystems, Foster City, CA, USA), Molecular Beacons (see, for example, Tyagi et al., Nature Biotechnology 14(3):303-8, 1996), Scorpions® (Molecular Probes Inc., Eugene, OR, USA)). In another example, a PCR product is detected by the binding of a fluorogenic dye that emits a fluorescent signal upon binding (e.g., SYBR® Green (Molecular Probes)). Such detection methods are useful for the detection of an ASPM PCR product. ,

In another embodiment, hybridization with PCR probes that are capable of detecting an ASPM nucleic acid molecule, including genomic sequences, or closely related molecules, may be used to hybridize to a nucleic acid sequence derived from a patient having a neoplasia. The specificity of the probe determines whether the probe hybridizes to a naturally occurring sequence, allelic variants, or other related sequences. Hybridization techniques may be used to identify mutations indicative of a neoplasia, or may be used to monitor expression levels of these genes (for example, by Northern analysis (Ausubel et al., supra). In yet another embodiment, humans may be diagnosed for a propensity to develop a neoplasia by direct analysis of the sequence of an ASPM nucleic acid molecule. The sequence of an ASPM nucleic acid molecule derived from a subject is compared to a reference sequence. An alteration in the sequence of the ASPM nucleic acid molecule relative to the reference indicates that the patient has or has a propensity to develop a neoplasia.

In another approach, diagnostic methods of the invention are used to assay the expression of an ASPM polypeptide in a biological sample relative to a reference (e.g., the level of ASPM polypeptide present in a corresponding control tissue). In one embodiment, the level of an ASPM polypeptide is detected using an antibody that specifically binds an ASPM polypeptide. Exemplary antibodies that specifically bind an ASPM polypeptide are described herein. Such antibodies are useful for the diagnosis of a neoplasia. Methods for measuring an antibody- ASPM complex include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index. Optical methods include microscopy (both confocal and non-confocal), imaging methods and non-imaging methods. Methods for performing these assays are readily known in the art. Useful assays include, for example, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot blot assay. These methods are also described in, e.g., Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991); and Harlow & Lane, supra. Immunoassays can be used to determine the quantity of ASPM in a sample, where an increase in the level of the ASPM polypeptide is diagnostic of a patient having a neoplasia.

In general, the measurement of an ASPM polypeptide or nucleic acid molecule in a subject sample is compared with a diagnostic amount present in a reference. A diagnostic amount distinguishes between a neoplastic tissue and a control tissue. The skilled artisan appreciates that the particular diagnostic amount used can be adjusted to increase sensitivity or specificity of the diagnostic assay depending on the preference of the diagnostician. In general, any significant increase (e.g., at least about 10%, 15%, 30%, 50%, 60%, 75%, 80%, or 90%) in the level of an ASPM polypeptide or nucleic acid molecule in the subject sample relative to a reference may be used to diagnose a neoplasia. In one embodiment, the reference is the level of ASPM polypeptide or nucleic acid molecule present in a control sample obtained from a patient that does not have a neoplasia. In another embodiment, the reference is a baseline level of ASPM present in a biologic sample derived from a patient prior to, during, or after treatment for a neoplasia. In yet another embodiment, the reference is a standardized curve.

Types of Biological Samples

The level of an ASPM polypeptide or nucleic acid molecule can be measured in different types of biologic samples. In one embodiment, the biologic sample is a tissue sample that includes cells of a tissue or organ. Such tissue is obtained, for example, from a biopsy. In another embodiment, the biologic sample is a biologic fluid sample (e.g., blood, blood plasma, serum, urine, seminal fluids, ascites, or cerebrospinal fluid).

Kits

The invention also provides kits for the diagnosis or monitoring of a neoplasia in a biological sample obtained from a subject. In one embodiment, the kit detects an increase in the expression of an ASPM nucleic acid molecule or polypeptide relative to a reference level of expression. In another embodiment, the kit detects an alteration in the sequence of an ASPM nucleic acid molecule derived from a subject relative to a reference sequence. In related embodiments, the kit includes reagents for monitoring the expression of an ASPM nucleic acid molecule, such as primers or probes that hybridize to an ASPM nucleic acid molecule. In other embodiments, the kit includes an antibody that binds to an ASPM polypeptide.

Optionally, the kit includes directions for monitoring ASPM nucleic acid molecule or polypeptide levels in a biological sample derived from a subject. In other embodiments, the kit comprises a sterile container which contains the primer, probe, antibody, or other detection regents; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container form known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding nucleic acids. The instructions will generally include information about the use of the primers or probes described herein and their use in diagnosing a neoplasia. Preferably, the kit further comprises any one or more of the reagents described in the diagnostic assays described herein. In other embodiments, the instructions include at least one of the following: description of the primer or probe; methods for using the enclosed materials for the diagnosis of a neoplasia; precautions; warnings; indications; clinical or research studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

Patient Monitoring

The disease state or treatment of a patient having a neoplasia can be monitored using the methods and compositions of the invention. In one embodiment, a microarray is used to assay the expression profile of an ASPM nucleic acid molecule. Such monitoring may be useful, for example, in assessing the efficacy of a particular drug in a patient. Therapeutics that alter the expression of an ASPM nucleic acid molecule or ASPM polypeptide (e.g., ASPM, ASPM variant 1, 2, or 3), are taken as particularly useful in the invention.

ASPM Antibodies

Antibodies are well known to those of ordinary skill in the science of immunology. As used herein, the term "antibody" means not only intact antibody molecules, but also fragments of antibody molecules that retain immunogen binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo. Accordingly, as used herein, the term "antibody" means not only intact immunoglobulin molecules but also the well-known active fragments F(ab')₂, and Fab. F(ab')₂, and Fab fragments which lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983). The antibodies of the invention comprise whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single chain V region fragments (scFv) and fusion polypeptides.

In one embodiment, an antibody that binds an ASPM polypeptide (e.g., ASPM, ASPM variant 1, 2, or 3) is monoclonal. Alternatively, the anti-ASPM antibody is a polyclonal antibody. The preparation and use of polyclonal antibodies are also known the skilled artisan. The invention also encompasses hybrid antibodies, in which one pair of heavy and light chains is obtained from a first antibody, while the other pair of heavy and light chains is obtained from a different second antibody. Such hybrids may also be formed using humanized heavy and light chains. Such antibodies are often referred to as "chimeric" antibodies.

In general, intact antibodies are said to contain "Fc" and "Fab" regions. The Fc regions are involved in complement activation and are not involved in antigen binding. An antibody from which the Fc' region has been enzymatically cleaved, or which has been produced without the Fc' region, designated an "F(ab')₂" fragment, retains both of the antigen binding sites of the intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an "Fab"' fragment, retains one of the antigen binding sites of the intact antibody. Fab' fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain, denoted "Fd." The Fd fragments are the major determinants of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity). Isolated Fd fragments retain the ability to specifically bind to immunogenic epitopes.

Antibodies can be made by any of the methods known in the art utilizing ASPM polypeptides (e.g., ASPM, ASPM variant 1, 2, or 3), or immunogenic fragments thereof, as an immunogen. One method of obtaining antibodies is to immunize suitable host animals with an immunogen and to follow standard procedures for polyclonal or monoclonal antibody production. The immunogen will facilitate presentation of the immunogen on the cell surface. Immunization of a suitable host can be carried out in a number of ways. Nucleic acid sequences encoding an ASPM polypeptide (e.g., ASPM, ASPM variant 1, 2, or 3), or immunogenic fragments thereof, can be provided to the host in a delivery vehicle that is taken up by immune cells of the host. The cells will in turn express the receptor on the cell surface generating an immunogenic response in the host. Alternatively, nucleic acid sequences encoding an ASPM polypeptide (e.g., ASPM, ASPM variant 1, 2, or 3), or immunogenic fragments thereof, can be expressed in cells in vitro, followed by isolation of the receptor and administration of the receptor to a suitable host in which antibodies are raised.

Using either approach, antibodies can then be purified from the host. Antibody purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column preferably run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and antiimmunoglobulin.

Antibodies can be conveniently produced from hybridoma cells engineered to express the antibody. Methods ofmaking hybridomas are well known in the art. The hybridoma cells can be cultured in a suitable medium, and spent medium can be used as an antibody source. Polynucleotides encoding the antibody of interest can in turn be obtained from the hybridoma that produces the antibody, and then the antibody may be produced synthetically or recombinantly from these DNA sequences. For the production of large amounts of antibody, it is generally more convenient to obtain an ascites fluid. The method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse. The mammal may be primed for ascites production by prior administration of a suitable composition; e.g., Pristane. Monoclonal antibodies (Mabs) produced by methods of the invention can be "humanized" by methods known in the art. "Humanized" antibodies are antibodies in which at least part of the sequence has been altered from its initial form to render it more like human immunoglobulins. Techniques to humanize antibodies are particularly useful when non- human animal (e.g., murine) antibodies are generated. Examples of methods for humanizing a murine antibody are provided in U.S. Patent Nos. 4,816,567, 5,530,101, 5,225,539, 5,585,089, 5,693,762 and 5,859,205. ASPM Polypeptide Expression

In general, ASPM polypeptides, variants, and fragments thereof may be produced by transformation of a suitable host cell with all or part of a polypeptide-encoding nucleic acid molecule or fragment thereof in a suitable expression vehicle. Those skilled in the field of molecular biology will understand that any of a wide variety of expression systems may be used to provide the recombinant protein. The precise host cell used is not critical to the invention. A polypeptide of the invention may be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., S£21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, or preferably COS cells). Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al., supra). The method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles may be chosen from those provided, e.g., in Cloning Vectors: A Laboratory Manual (P. H. Pouwels et al., 1985, Supp. 1987).

A variety of expression systems exist for the production of the polypeptides of the invention. Expression vectors useful for producing such polypeptides include, without limitation, chromosomal, episomal, and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof.

One particular bacterial expression system for polypeptide production is the E. coli pET expression system (Novagen, Inc., Madison, Wis). According to this expression system, DNA encoding a polypeptide is inserted into a pET vector in an orientation designed to allow expression. Since the gene encoding such a polypeptide is under the control of the T7 regulatory signals, expression of the polypeptide is achieved by inducing the expression of T7 RNA polymerase in the host cell. This is typically achieved using host strains that express T7 RNA polymerase in response to IPTG induction. Once produced, recombinant polypeptide is then isolated according to standard methods known in the art, for example, those described herein.

Another bacterial expression system for polypeptide production is the pGEX expression system (Pharmacia). This system employs a GST gene fusion system that is designed for high-level expression of genes or gene fragments as fusion proteins with rapid purification and recovery of functional gene products. The protein of interest is fused to the carboxyl terminus of the glutathione S-transferase protein from Schistosoma japonicum and is readily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. Fusion proteins can be recovered under mild conditions by elution with glutathione. Cleavage of the glutathione S-transferase domain from the fusion protein is facilitated by the presence of recognition sites for site-specific proteases upstream of this domain. For example, proteins expressed in pGEX-2T plasmids may be cleaved with thrombin; those expressed in pGEX-3X may be cleaved with factor Xa.

Once the recombinant polypeptide of the invention is expressed, it is isolated, e.g., using affinity chromatography. In one example, an antibody (e.g., produced as described herein) raised against a polypeptide of the invention may be attached to a column and used to isolate the recombinant polypeptide. Lysis and fractionation of polypeptide-harboring cells prior to affinity chromatography may be performed by standard methods (see, e.g., Ausubel et al., supra). Once isolated, the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980). Polypeptides of the invention, particularly short peptide fragments, can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford, 111.). These general techniques of polypeptide expression and purification can also be used to produce and isolate useful peptide fragments or analogs (described herein).

ASPM Polypeptides and Analogs Also included in the invention are ASPM polypeptides, variants, or fragments thereof containing at least one alteration relative to a reference sequence. Such alterations include certain mutations, deletions, insertions, or post-translational modifications. The invention further includes analogs of any naturally-occurring polypeptide of the invention. Analogs can differ from naturally-occurring polypeptides of the invention by amino acid sequence differences, by post-translational modifications, or by both. Analogs of the invention will generally exhibit at least 85%, more preferably 90%, and most preferably 95% or even 99% identity with all or part of a naturally-occurring amino acid sequence of the invention. The length of sequence comparison is at least 10, 13, 15 amino acid residues, preferably at least 25 amino acid residues, and more preferably more than 35 amino acid residues. Again, in an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e^"3 and e^"100 indicating a closely related sequence. Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes. Analogs can also differ from the naturally-occurring polypeptides of the invention by alterations in primary sequence. These include genetic variants, both natural and induced (for example, resulting from random mutagenesis by irradiation or exposure to ethanemethylsulfate or by site-specific mutagenesis as described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual (2d ed.), CSH Press, 1989, or Ausubel et al., supra). Also included are cyclized peptides, molecules, and analogs which contain residues other than L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids.

In addition to full-length polypeptides, the invention also includes fragments of any one of the polypeptides of the invention. As used herein, the term "a fragment" means at least 5, 10, 13, or 15 amino acids. In other embodiments a fragment is at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids, and in other embodiments at least 60 to 80 or more contiguous amino acids. Fragments of the invention can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).

ASPM Polynucleotides In general, the invention includes any nucleic acid sequence encoding an ASPM polypeptide (e.g., ASPM, ASPM variant 1, 2, or 3). Also included in the methods of the invention are any nucleic acid molecule containing at least one strand that hybridizes with such a nucleic acid sequence (e.g., an inhibitory nucleic acid molecule, such as a dsRNA, siRNA, shRNA, or antisense molecule). An isolated nucleic acid molecule can be manipulated using recombinant DNA techniques well known in the art. Thus, a nucleotide sequence contained in a vector in which 5' and 3' restriction sites are known, or for which polymerase chain reaction (PCR) primer sequences have been disclosed, is considered isolated, but a nucleic acid sequence existing in its native state in its natural host is not. An isolated nucleic acid may be substantially purified, but need not be. For example, a nucleic acid molecule that is isolated within a cloning or expression vector may comprise only a tiny percentage of the material in the cell in which it resides. Such a nucleic acid is isolated, however, as the term is used herein, because it can be manipulated using standard techniques known to those of ordinary skill in the art.

ASPM Polynucleotide Therapy

Polynucleotide therapy featuring a polynucleotide encoding an ASPM protein, variant, or fragment thereof is another therapeutic approach for treating a neoplasia. Such nucleic acid molecules can be delivered to cells of a subject having a neoplasia. The nucleic acid molecules must be delivered to the cells of a subject in a form in which they can be taken up so that therapeutically effective levels of an ASPM protein (e.g., ASPM, ASPM variant 1, 2, or 3) or fragment thereof can be produced.

Transducing viral (e.g., retroviral, adenoviral, and adeno-associated viral) vectors can be used for somatic cell gene therapy, especially because of their high efficiency of infection and stable integration and expression (see, e.g., Cayouette et al., Human Gene Therapy 8:423-430, 1997; Kido et al., Current Eye Research 15:833-844, 1996; Bloomer et al., Journal of Virology 71 :6641-6649, 1997; Naldini et al., Science 272:263-267, 1996; and Miyoshi et al., Proc. Natl. Acad. Sci. U.S.A. 94:10319, 1997). For example, a polynucleotide encoding an ASPM protein, variant, or a fragment thereof, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus (also see, for example, the vectors of Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244:1275-1281, 1989; Eglitis et al., BioTechniques 6:608-614, 1988; Tolstoshev et al., Current Opinion in Biotechnology 1:55- 61, 1990; Sharp, The Lancet 337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409, 1984; Moen, Blood Cells 17:407-416, 1991; Miller et al., Biotechnology 7:980-990, 1989; Le Gal La Salle et al., Science 259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995). Retroviral vectors are particularly well developed and have been used in clinical settings (Rosenberg et al., N. Engl. J. Med 323:370, 1990; Anderson et al., U.S. Pat. No. 5,399,346). Most preferably, a viral vector is used to administer an ASPM polynucleotide systemically.

Non- viral approaches can also be employed for the introduction of therapeutic to a cell of a patient diagnosed as having a neoplasia. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection (Feigner et al., Proc. Natl. Acad. Sci. U.S.A. 84:7413, 1987; Ono et al., Neuroscience Letters 17:259, 1990; Brigham et al., Am. J. Med. Sci. 298:278, 1989; Staubinger et al., Methods in Enzymology 101:512, 1983), asialoorosomucoid-polylysine conjugation (Wu et al., Journal of Biological Chemistry 263:14621, 1988; Wu et al., Journal of Biological Chemistry 264:16985, 1989), or by micro-injection under surgical conditions (Wolff et al., Science 247: 1465, 1990). Preferably the nucleic acids are administered in combination with a liposome and protamine.

Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a patient can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue. cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.

Another therapeutic approach included in the invention involves administration of a recombinant therapeutic, such as a recombinant ASPM protein, variant, or fragment thereof, either directly to the site of a potential or actual disease-affected tissue or systemically (for example, by any conventional recombinant protein administration technique). The dosage of the administered protein depends on a number of factors, including the size and health of the individual patient. For any particular subject, the specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions. Screening Assays

As reported herein, the expression of an ASPM polypeptide is increased in neoplastic tissues. Accordingly, compounds that modulate the expression or activity of an ASPM polypeptide, variant, or fragment thereof are useful in the methods of the invention for the treatment or prevention of a neoplasm (e.g., breast, colon, lymph, ovary, stomach, thyroid, testis, and uterine cancer). Any number of methods are available for carrying out screening assays to identify such compounds. In one approach, candidate compounds are identified that specifically bind to and alter the activity of a polypeptide of the invention (e.g., an ASPM activity associated with cell proliferation, mitosis or maintenance of chromosomal stability). Methods of assaying such biological activities are known in the art and are described herein. The efficacy of such a candidate compound is dependent upon its ability to interact with an ASPM polypeptide, variant, or fragment. Such an interaction can be readily assayed using any number of standard binding techniques and functional assays (e.g., those described in Ausubel et al., supra). For example, a candidate compound may be tested in vitro for interaction and binding with a polypeptide of the invention and its ability to modulate cell proliferation, mitosis, or maintenance of chromosomal stability. ASPM's function in chromosomal stability can be assayed by detecting, for example, chromosomal nondysjunction in cells where endogenous ASPM expression is perturbed or reduced. Standard methods for perturbing or reducing ASPM expression include mutating or deleting an endogenous ASPM sequence, interfering with ASPM expression using RNAi, or microinjecting an ASPM-expressing cell with an antibody that binds ASPM and interferes with its function. Alternatively, chromosomal nondysjunction can be assayed in vivo, for example, in a mouse model in which ASPM has been knocked out by homologous recombination, or any other standard method. Potential agonists and antagonists of an ASPM polypeptide include organic molecules, peptides, peptide mimetics, polypeptides, nucleic acid molecules (e.g., double- stranded RNAs, siRNAs, antisense polynucleotides), and antibodies that bind to a nucleic acid sequence or polypeptide of the invention and thereby inhibit or extinguish its activity. Potential antagonists also include small molecules that bind to the ASPM polypeptide thereby preventing binding to cellular molecules with which the ASPM polypeptide normally interacts, such that the normal biological activity of the ASPM polypeptide is reduced or inhibited. Small molecules of the invention preferably have a molecular weight below 2,000 daltons, more preferably between 300 and 1,000 daltons, and most preferably between 400 and 700 daltons. It is preferred that these small molecules are organic molecules. In one particular example, a candidate compound that binds to an ASPM polypeptide, variant, or fragment thereof may be identified using a chromatography-based technique. For example, a recombinant polypeptide of the invention may be purified by standard techniques from cells engineered to express the polypeptide (e.g., those described above) and may be immobilized on a column. A solution of candidate compounds is then passed through the column, and a compound specific for the ASPM polypeptide is identified on the basis of its ability to bind to the ASPM polypeptide and be immobilized on the column. To isolate the compound, the column is washed to remove non-specifically bound molecules, and the compound of interest is then released from the column and collected. Similar methods may be used to isolate a compound bound to a polypeptide microarray. Compounds isolated by this method (or any other appropriate method) may, if desired, be further purified (e.g., by high performance liquid chromatography). In addition, these candidate compounds may be tested for their ability to alter the biological activity of an ASPM polypeptide (e.g., ASPM, ASPM variant 1, 2, or 3). Compounds that are identified as binding to a polypeptide of the invention with an affinity constant less than or equal to 10 mM are considered particularly useful in the invention. Alternatively, any in vivo protein interaction detection system, for example, any two-hybrid assay may be utilized to identify compounds that interact with an ASPM polypeptide. Interacting compounds isolated by this method (or any other appropriate , method) may, if desired, be further purified (e.g., by high performance liquid chromatography). Compounds isolated by any approach described herein may be used as therapeutics to treat a neoplasia in a human patient.

In addition, compounds that inhibit the expression of an ASPM nucleic acid molecule whose expression is increased in a patient having a neoplasia are also useful in the methods of the invention. Any number of methods are available for carrying out screening assays to identify new candidate compounds that alter the expression of an ASPM nucleic acid molecule. In one working example, candidate compounds are added at varying concentrations to the culture medium of cultured cells expressing one of the nucleic acid sequences of the invention. Gene expression is then measured, for example, by microarray analysis, Northern blot analysis (Ausubel et al., supra), or RT-PCR, using any appropriate fragment prepared from the nucleic acid molecule as a hybridization probe. The level of gene expression in the presence of the candidate compound is compared to the level measured in a control culture medium lacking the candidate molecule. A compound that promotes an alteration in the expression of an ASPM gene, or a functional equivalent thereof, is considered useful in the invention; such a molecule may be used, for example, as a therapeutic to treat a neoplasia in a human patient.

In another approach, the effect of candidate compounds is measured at the level of polypeptide production to identify those that promote an alteration in an ASPM polypeptide level. The level of ASPM polypeptide can be assayed using any standard method. Standard immunological techniques include Western blotting or immunoprecipitation with an antibody specific for an ASPM polypeptide (e.g., ASPM, ASPM variant 1, 2, or 3). For example, immunoassays may be used to detect or monitor the expression of at least one of the polypeptides of the invention in an organism. Polyclonal or monoclonal antibodies (produced as described above) that are capable of binding to such a polypeptide may be used in any standard immunoassay format (e.g., ELISA, Western blot, or RIA assay) to measure the level of the polypeptide. In some embodiments, a compound that promotes a decrease in the expression or biological activity of the polypeptide is considered particularly useful. Again, such a molecule may be used, for example, as a therapeutic to delay, ameliorate, or treat a neoplasia in a human patient.

In another embodiment, a nucleic acid described herein (e.g., an ASPM nucleic acid) is expressed as a transcriptional or translational fusion with a detectable reporter, and expressed in an isolated cell (e.g., mammalian or insect cell) under the control of a heterologous promoter, such as an inducible promoter. The cell expressing the fusion protein is then contacted with a candidate compound, and the expression of the detectable reporter in that cell is compared to the expression of the detectable reporter in an untreated control cell. A candidate compound that alters the expression of the detectable reporter is a compound that is useful for the treatment of a neoplasia. In one embodiment, the compound decreases the expression of the reporter. Each of the DNA sequences listed herein may also be used in the discovery and development of a therapeutic compound for the treatment of neoplasia. The encoded protein, upon expression, can be used as a target for the screening of drugs. Additionally, the DNA sequences encoding the amino terminal regions of the encoded protein or Shine-Delgarno or other translation facilitating sequences of the respective mRNA can be used to construct sequences that promote the expression of the coding sequence of interest. Such sequences may be isolated by standard techniques (Ausubel et al., supra).

The invention also includes novel compounds identified by the above-described screening assays. Optionally, such compounds are characterized in one or more appropriate animal models to determine the efficacy of the compound for the treatment of a neoplasia. Desirably, characterization in an animal model can also be used to determine the toxicity, side effects, or mechanism of action of treatment with such a compound. Furthermore, novel compounds identified in any of the above-described screening assays may be used for the treatment of a neoplasia in a subject. Such compounds are useful alone or in combination with other conventional therapies known in the art.

Test Compounds and Extracts

In general, compounds capable of inhibiting the growth or proliferation of a neoplasia by altering the expression or biological activity of an ASPM polypeptide, variant, or fragment thereof are identified from large libraries of either natural product or synthetic (or semisynthetic) extracts or chemical libraries according to methods known in the art. Numerous methods are also available for generating random or directed synthesis (e.g., semi-synthesis or total synthesis) of any number of chemical compounds, including, but not limited to, saccharide-, lipid-, peptide-, and nucleic acid-based compounds. Synthetic compound libraries are commercially available from Brandon Associates (Merrimack, N.H.) and Aldrich Chemical (Milwaukee, Wis.). Alternatively, libraries of natural compounds in the form of ^" bacterial, fungal, plant, and animal extracts are commercially available from a number of sources, including Biotics (Sussex, UK), Xenova (Slough, UK), Harbor Branch Oceangraphics Institute (Ft. Pierce, FIa.), and PharmaMar, U.S.A. (Cambridge, Mass.). In one embodiment, test compounds of the invention are present in any combinatorial library known in the art, including: biological libraries; peptoid libraries (libraries of molecules having the functionalities of peptides, but with a novel, non-peptide backbone which are resistant to enzymatic degradation but which nevertheless remain bioactive; see, e.g., Zuckermann, R.N. et al, J. Med. Chem. 37:2678-85, 1994); spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one-bead one-compound¹ library method; and synthetic library methods using affinity chromatography selection. The biological library and peptoid library approaches are limited to peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer DrugDes. 12:145, 1997).

Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al, Proc. Natl. Acad. ScL U.S.A. 90:6909, 1993; Erb et al, Proc. Natl. Acad. ScL USA 91:11422, 1994; Zuckermann et al, J. Med. Chem. 37:2678, 1994; Cho et al, Science 261:1303, 1993; Carrell et al, Angew. Chem. Int. Ed. Engl. 33:2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33:2061, 1994; and Gallop et al, J. Med. Client.

37:1233, 1994.

Libraries of compounds may be presented in solution (e.g., Houghten, Biotechniques

13:412-421, 1992), or on beads (Lam, Nature 354:82-84, 1991), chips (Fodor, Nature 364:555-556, 1993), bacteria (Ladner, U.S. Patent No. 5,223,409), spores (Ladner U.S. Patent

No. 5,223,409), plasmids (Cull et al, Proc Natl Acad Sd USA 89:1865-1869, 1992) or on phage (Scott and Smith, Science 249:386-390, 1990; Devlin, Science 249:404-406, 1990;

Cwirla et al. Proc. Natl. Acad. Sd. 87:6378-6382, 1990; Felici, J. MoI. Biol. 222:301-310,

1991; Ladner supra.). In addition, those skilled in the art of drug discovery and development readily understand that methods for dereplication (e.g., taxonomic dereplication, biological dereplication, and chemical dereplication, or any combination thereof) or the elimination of replicates or repeats of materials already known for their anti-neoplastic activity should be employed whenever possible. Those skilled in the field of drug discovery and development will understand that the precise source of a compound or test extract is not critical to the screening procedure(s) of the invention. Accordingly, virtually any number of chemical extracts or compounds can be screened using the methods described herein. Examples of such extracts or compounds include, but are not limited to, plant-, fungal-, prokaryotic- or animal-based extracts, fermentation broths, and synthetic compounds, as well as modification of existing compounds.

When a crude extract is found to alter the biological activity of an ASPM polypeptide, variant, or fragment thereof, further fractionation of the positive lead extract is necessary to isolate chemical constituents responsible for the observed effect. Thus, the goal of the extraction, fractionation, and purification process is the careful characterization and identification of a chemical entity within the crude extract having anti-neoplastic activity.

Methods of fractionation and purification of such heterogenous extracts are known in the art.

If desired, compounds shown to be useful agents for the treatment of a neoplasm are chemically modified according to methods known in the art.

Methods of Assaying ASPM Biological Activity

Therapeutics useful in the methods of the invention include, but are not limited to, those that have an anti-neoplastic activity or those that alter an ASPM biological activity associated with cell proliferation or maintenance of chromosomal stability. Neoplastic cell growth is not subject to the same regulatory mechanisms that govern the growth or proliferation of normal cells. Compounds that reduce the growth or proliferation of a neoplasm are useful for the treatment of neoplasms. Methods of assaying cell growth and proliferation are known in the art. See, for example, Kittler et al. (Nature. 432 (7020): 1036- 40, 2004) and by Miyamoto et al. (Nature 416(6883):865-9, 2002). Assays for cell proliferation generally involve the measurement of DNA synthesis during cell replication. In one embodiment, DNA synthesis is detected using labeled DNA precursors, such as ([³H]- Thymidine or 5-bromo-2'-deoxyuridine [BrdU], which are added to cells (or animals) and then the incorporation of these precursors into genomic DNA during the S phase of the cell cycle (replication) is detected (Ruefli-Brasse et al., Science 302(5650):1581-4, 2003; Gu et al., Science 302 (5644):445-9, 2003).

Candidate compounds that reduce the survival of a neoplastic cell are also useful as anti-neoplasm therapeutics. Assays for measuring cell viability are known in the art, and are described, for example, by Crouch et al. (J. Immunol. Meth. 160, 81-8); Kangas et al. (Med. Biol.62, 338-43, 1984); Lundin et al., (Meth. Enzymol.133, 27-42, 1986); Petty et al.

(Comparison of J. Biolum. Chemilum.lO, 29-34, 1995); and Cree et al. (Anticancer Drugs 6: 398-404, 1995). Cell viability can be assayed using a variety of methods, including MTT (3- (4,5-dimethylthiazolyl)-2,5-diphenyltetrazolium bromide) (Barltrop, Bioorg. & Med. Chem. Lett.1: 611, 1991; Cory et al., Cancer Comm. 3, 207-12, 1991; Paull J. Heterocyclic Chem. 25, 911, 1988). Assays for cell viability are also available commercially. These assays include CELLTITER-GLO^® Luminescent Cell Viability Assay (Promega), which uses luciferase technology to detect ATP and quantify the health or number of cells in culture, and the CellTiter-Glo^® Luminescent Cell Viability Assay, which is a lactate dehyrodgenase (LDH) cytotoxicity assay. Candidate compounds that increase neoplastic cell death (e.g., increase apoptosis) are also useful as anti-neoplasm therapeutics. Assays for measuring cell apoptosis are known to the skilled artisan. Apoptotic cells are characterized by characteristic morphological changes, including chromatin condensation, cell shrinkage and membrane blebbing, which can be clearly observed using light microscopy. The biochemical features of apoptosis include DNA fragmentation, protein cleavage at specific locations, increased mitochondrial membrane permeability, and the appearance of phosphatidylserine on the cell membrane surface. Assays for apoptosis are known in the art. Exemplary assays include TUNEL (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling) assays, caspase activity (specifically caspase-3) assays, and assays for fas-ligand and annexin V. Commercially available products for detecting apoptosis include, for example, Apo-ONE^® Homogeneous Caspase-3/7 Assay, FragEL TUNEL kit (ONCOGENE RESEARCH PRODUCTS, San Diego, CA)₅ the ApoBrdU DNA Fragmentation Assay (BIOVISION, Mountain View, CA), and the Quick Apoptotic DNA Ladder Detection Kit (BIOVTSION, Mountain View, CA).

Microarrays

The methods of the invention may also be used for microarray-based assays that provide for the high-throughput analysis of cancer biomarkers. The ASPM nucleic acid molecules or polypeptides of the invention are useful as hybridizable array elements in such a microarray. The array elements are organized in an ordered fashion such that each element is present at a specified location on the substrate. Useful substrate materials include membranes, composed of paper, nylon or other materials, filters, chips, glass slides, and other solid supports. The ordered arrangement of the array elements allows hybridization patterns and intensities to be interpreted as expression levels of particular genes or proteins. Methods for making nucleic acid microarrays are known to the skilled artisan and are described, for example, in U.S. Pat. No. 5,837,832, Lockhart, et al. (Nat. Biotech. 14:1675-1680, 1996), and Schena, et al. (Proc. Natl. Acad. Sci. 93:10614-10619, 1996), herein incorporated by reference. Methods for making polypeptide microarrays are described, for example, by Ge (Nucleic Acids Res. 28:e3.i-e3.vii, 2000), MacBeath et al., (Science 289:1760-1763, 2000), Zhu et al. (Nature Genet. 26:283-289), and in U.S. Pat. No. 6,436,665, hereby incorporated by reference.

Nucleic Acid Microarrays

To produce a nucleic acid microarray oligonucleotides may be synthesized or bound to the surface of a substrate using a chemical coupling procedure and an ink jet application apparatus, as described in PCT application WO95/251116 (Baldeschweiler et al.), incorporated herein by reference. Alternatively, a gridded array may be used to arrange and link cDNA fragments or oligonucleotides to the surface of a substrate using a vacuum system, thermal, UV, mechanical or chemical bonding procedure.

A nucleic acid molecule (e.g. RNA or DNA) derived from a biological sample may be used to produce a hybridization probe as described herein. The biological samples are generally derived from a patient, preferably as a bodily fluid (such as blood, cerebrospinal fluid, phlegm, saliva, or urine) or tissue sample (e.g. a tissue sample obtained by biopsy). For some applications, cultured cells or other tissue preparations may be used. The mRNA is isolated according to standard methods, and cDNA is produced and used as a template to make complementary RNA suitable for hybridization. Such methods are described herein. The RNA is amplified in the presence of fluorescent nucleotides, and the labeled probes are then incubated with the microarray to allow the probe sequence to hybridize to complementary oligonucleotides (e.g., ASPM nucleic acid molecules) bound to the microarray.

Incubation conditions are adjusted such that hybridization occurs with precise complementary matches or with various degrees of less complementarity depending on the degree of stringency employed. For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30⁰C, more preferably of at least about 37°C, and most preferably of at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In one embodiment, hybridization will occur at 3O⁰C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In another embodiment, hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In yet another embodiment, hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

The removal of nonhybridized probes may be accomplished, for example, by washing. The washing steps that follow hybridization can also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash • steps will ordinarily include a temperature of at least about 25°C, at least about 42°C, or at least about 68⁰C. In one embodiment, wash steps will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another embodiment, wash steps will occur at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In yet another embodiment, wash steps will occur at 68°C in 15 mMNaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. A detection system may be used to measure the absence, presence, and amount of hybridization for all of the distinct sequences simultaneously (e.g., Heller et al., Proc. Natl. Acad. Sci. 94:2150-2155, 1997). Preferably, a scanner is used to determine the levels and patterns of fluorescence.

Protein Microarrays ASPM polypeptides (e.g., ASPM, ASPM variant 1, 2, or 3), such as those described herein, may also be analyzed using protein microarrays. Such arrays are useful in high- throughput low-cost screens to identify peptide or candidate compounds that bind a polypeptide of the invention, or fragment thereof. Typically, protein microarrays feature a protein, or fragment thereof, bound to a solid support. Suitable solid supports include membranes (e.g., membranes composed of nitrocellulose, paper, or other material), polymer- based films (e.g., polystyrene), beads, or glass slides. For some applications, ASPM polypeptides (e.g., ASPM, ASPM variant 1, 2, or 3) are spotted on a substrate using any convenient method known to the skilled artisan (e.g., by hand or by inkjet printer). Preferably, such methods retain the biological activity or function of the protein bound to the substrate (e.g., ASPM antibody binding).

The protein microarray is hybridized with a detectable probe. Such probes can be polypeptide (e.g., an ASPM antibody), nucleic acid, or small molecules. For some applications, polypeptide and nucleic acid probes are derived from a biological sample taken from a patient, such as a bodily fluid (such as blood, urine, saliva, or phlegm); a homogenized tissue sample (e.g. a tissue sample obtained by biopsy); or cultured cells (e.g., lymphocytes). Probes can also include antibodies, candidate peptides, nucleic acids, or small molecule compounds derived from a peptide, nucleic acid, or chemical library. Hybridization conditions (e.g., temperature, pH, protein concentration, and ionic strength) are optimized to promote specific interactions. Such conditions are known to the skilled artisan and are described, for example, in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual. 1998, New York: Cold Spring Harbor Laboratories. After removal of non-specific probes, specifically bound probes are detected, for example, by fluorescence, enzyme activity (e.g., an enzyme-linked calorimetric assay), direct immunoassay, radiometric assay, or any other suitable detectable method known to the skilled artisan. Detection of an increase in the amount of an ASPM polypeptide (e.g., ASPM, ASPM variant 1, 2, or 3) or an ASPM polynucleotide present in a patient sample is useful as a diagnostic for the presence of a neoplasia. Optionally, ASPM detection may be combined with the detection of other biomarkers, where the presence or level of the biomarker is correlated with the presence of a neoplasia.

Pharmaceutical Compositions

The present invention contemplates pharmaceutical preparations comprising an ASPM protein, a polynucleotide that encodes an ASPM protein, or an ASPM inhibitory nucleic acid molecule (e.g., a polynucleotide that hybridizes to and interferes with the expression of an ASPM polynucleotide), together with a pharmaceutically acceptable carrier. Polynucleotides of the invention may be administered as part of a pharmaceutical composition. The compositions should be sterile and contain a therapeutically effective amount of the polypeptides or nucleic acid molecules in a unit of weight or volume suitable for administration to a subject.

These compositions ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution. As an example of a lyophilized formulation, 10 mL vials are filled with 5 mL of sterile-filtered 1% (w/v) aqueous ASPM polynucleotide solution, such as an aqueous solution of ASPM polynucleotide or polypeptide, and the resulting mixture can then be lyophilized. The infusion solution can be prepared by reconstituting the lyophilized material using sterile Water-for-Injection (WFI).

The ASPM polynucleotide, or polypeptide, or analogs may be combined, optionally, with a pharmaceutically acceptable excipient. The term "pharmaceutically-acceptable excipient" as used herein means one or more compatible solid or liquid filler, diluents or encapsulating substances that are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate administration. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction that would substantially impair the desired pharmaceutical efficacy.

The compositions can be administered in effective amounts. The effective amount will depend upon the mode of administration, the particular condition being treated and the desired outcome. It may also depend upon the stage of the condition, the age and physical condition of the subject, the nature of concurrent therapy, if any, and like factors well known to the medical practitioner. For therapeutic applications, it is that amount sufficient to achieve a medically desirable result.

With respect to a subject having an neoplastic disease or disorder, an effective amount is sufficient to stabilize, slow, or reduce the proliferation of the neoplasm. Generally, doses of active polynucleotide compositions of the present invention would be from about 0.01 mg/kg per day to about 1000 mg/kg per day. It is expected that doses ranging from about 50 to about 2000 mg/kg will be suitable. Lower doses will result from certain forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day are contemplated to achieve appropriate systemic levels of the ASPM polynucleotide or polypeptide compositions of the present invention.

A variety of administration routes are available. The methods of the invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Other modes of administration include oral, rectal, topical, intraocular, buccal, intravaginal, intracisternal, intracerebroventricular, intratracheal, nasal, transdermal, within/on implants, e.g., fibers such as collagen, osmotic pumps, or grafts comprising appropriately transformed cells, etc., or parenteral routes. A particular method of administration involves coating, embedding or derivatizing fibers, such as collagen fibers, protein polymers, etc. with therapeutic proteins. Other useful approaches are described in Otto, D. et al., J. Neurosci. Res. 22: 83-91 and in Otto, D. and Unsicker, K. J. Neurosci. 10: 1912-1921.

Combination Therapies for the Treatment of a Neoplasm

Compositions and methods of the invention may be used in combination with any conventional therapy known in the art. In one embodiment, an ASPM polynucleotide or polypeptide composition of the invention having anti-neoplastic activity may be used in combination with any anti-neoplastic therapy known in the art. Exemplary anti-neoplastic therapies include, for example, chemotherapy, cryotherapy, hormone therapy, radiotherapy, and surgery. A ASPM polynucleotide composition of the invention may, if desired, include one or more chemotherapeutics typically used in the treatment of a neoplasm, such as abiraterone acetate, altretamine, anhydrovinblastine, auristatin, bexarotene, bicalutamide, BMSl 84476, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide, bleomycin, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly- l-Lproline-t- butylamide, cachectin, cemadotin, chlorambucil, cyclophosphamide, 3',4'-didehydro-4'- deoxy-δ'-norvin- caleukoblastine, docetaxol, doxetaxel, cyclophosphamide, carboplatin, carmustine (BCNU),cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, daunorubicin, dolastatin, doxorubicin (adriamycin), etoposide, 5- fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyureataxanes, ifosfamide, liarozole, lonidamine, lomustine (CCNU), mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, 5- fluorouracil, nilutamide, onapristone, paclitaxel, prednimustine, procarbazine, RPR109881, stramustine phosphate, tamoxifen, tasonermin, taxol, tretinoin, vinblastine, vincristine, vindesine sulfate, and vinflunine. Other examples of chemotherapeutic agents can be found in Cancer Principles and Practice of Oncology by V. T. Devita and S. Hellman (editors), 6th edition (Feb. 15, 2001), Lippincott Williams & Wilkins Publishers.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

ASPM and Microcephaly

Primary autosomal recessive microcephaly is a genetic disorder in which an affected individual has a head circumference >3 standard deviations below the age- and sex-related mean and is mentally retarded, but typically has no other abnormal findings. Brain scans show that while the entire brain is reduced in size, the cerebral cortex is most severely affected. Because the vast majority of neurons are generated by week 21 of fetal life, microcephaly is likely due to a reduced production of neurons. The most common cause of microcephaly appears to be mutations in the ASPM gene.

The ASPM gene, which spans 65 kb of genomic DNA at Iq31, encodes a 10,434 base pair coding sequence (GenBank Accession No. NM_018136) containing 28 exons.

Expression studies in mice show ϊhatAspm is specifically expressed in the cerebral cortical •ventricular zone, the proliferative region of the lateral and medial ganglionic eminence, and the ventricular zone of the dorsal diencephalon at embryonic day 14.5. Expression diminishes by E16.5 and is greatly reduced at birth. Expression after birth was limited to regions of the brain that showed persistent postnatal neurogenesis. Thus, the expression pattern oϊAspm suggests that it has an important role in regulating neurogenesis before and after birth.

The predicted ASPM protein has two distinguished regions, a tandem pair of N- terminal putative calponin-homology (CH) domains and a large block of IQ motifs (the single letter codes for the amino acids isoleucine (I) and glutamine (Q)), a sequence which mediates interactions with calmodulin and calmodulin-related proteins. CH domains are about 100 residues long and are commonly involved in actin binding, but many other substrates are found in vivo (Gimona et al., FEBS Lett. 513, 98-106, 2002; Korenbaum et al., J. Cell Sci. 115, 3543-3545, 2002). The IQ calmodulin-binding motif comprises 20-25 amino acids, with the core fitting the consensus IQXXXRGXXXR (where X is any amino acid) (Mooseker et al., Annual. Rev. Cell. Dev. Biol. 11, 633-675, 1995; Houdusse et al., Structure 4, 1475-1490, 1996). CH and IQ domains were first discovered in motor proteins such as unconventional myosins (Matin et al., Protein Sci. 12, 2909-2923). Calmodulin binding to IQ-motifs induces a conformational change in the proteins that regulates the binding of actin to the amino- terminal CH domains (Bahler et al., FEBS Lett. 513, 107-113). Typically unconventional myosins contain between 2 and 6 IQ motifs. They are not identical within a single protein, i.e. some of them have a higher affinity for the Ca⁺² -free form of calmodulin, while other motifs have a higher affinity for the Ca⁺²/calmodulin. The number of IQ motifs and their divergence seems to determine the length of the lever arm and hence the step-size of myosin motor.

In contrast to unconventional myosins, ASPM does not have the catalytic Sl motor domain and therefore it cannot function as a mechanochemical protein. ASPM also differs from all known motor proteins by an extreme abundance of IQ motifs. The protein contains 81 putative calmodulin-binding motifs, most of which are encoded by exon 18, the largest exon in ASPM as reported herein.

The predicted ASPM protein is conserved between mammals, Drosophila, and worm, with a consistent correlation between nervous system complexity and protein length, principally involving an increase in the number of encoded IQ domains. For example, the Caenorhabditis elegans ASP homologue of ASPM contains 2 IQ domains; the Drosophila melonagaster ASP protein contains 24 repeats, and there are between 60-81 IQ domains in mammals. An interspecies sequence comparison reveals that the most highly diverged regions between primates and non-primate mammals are concentrated within the IQ array, suggesting that selection of specific segments of the ASPM gene may play a significant role in brain evolution of mammals (Zhang et al., Genetics 165, 2063-2070, 2003; Evans et al., Hum. MoI. Genet. 13, 489-494, 2004; Kouprina et al., PIoS Biol. 5, E126, 2004).

Drosophila abnormal spindle (αspj-deficient mutants exhibit a mitotic metaphase checkpoint arrest with abnormal spindle poles, suggesting that Asp is required for the integrity of microtubule organizing centers. The Drosophila asp gene encodes a 220 kDa microtubule-associated protein found at the spindle poles and centrosomes from prophase to early telophase. Besides calmodulin-IQ-binding and calponin homology domains, the Asp protein has consensus phosphorylation sites for CDKl and MAP kinases (Saunders et al., J. Cell. Biol. 137, 881-890, 1997). Asp is co-purified with γ — tubulin from centrosomes and they are both required for the organization of microtubules into asters (Avides et al., Science 283, 1733-1735, 1999). This activity is dependent on the phosphorylation of Asp by the kinase Polo (Avides et al., Nat. Cell. Biol. 4, 421-424, 2001; Glover et al., Oncogene 24, 230-237, 2005). Together these observations suggest that mutations in human ASPM may cause microcephaly due to the disregulation of mitotic spindle activity in neuronal progenitor cells.

As reported in the Examples below, present studies now indicate that ASPM and alternatively spliced isoforms of ASPM are expressed in a variety of embryonic and adult tissues and are upregulated in neoplasia.

Example 1: ASPM is expressed in embryonic and adult tissues

Using in situ hybridization, previous studies detected expression of mouse ASPM in the cerebral cortical ventricular zone, the proliferative region of the lateral and medial ganglionic eminence and the ventricular zone o the dorsal diencephalon (Bond et al., Nat. Genet. 32, 316-320, 2005; Luers et al., Meek Dev. 118, 229-232, 2002). The present study investigated gene expression in the whole mouse embryo and found that Aspm is also expressed in during the development of a variety of organs, including the liver, heart, lung, and kidney (Figure IA). Similar results were obtained for human ASPM using RT-PCR analysis. Human ASPM transcripts were detected in a variety of different human embryonic tissues, including brain, bladder, colon, heart, liver, lung, skeletal muscle, skin, spleen, and stomach. Human expression was analyzed using two pairs of primers specific to exons 2 and 3, and exons 13 and 14 (FN2-3/RN2-3 and FN14-15/RN14-15, respectively). The nucleotide sequences of primers used in this work are provided at Table 1 (below) (SEQ ID Nos:98- 117). Table !: Primers

Oligonucleotide name Sequence Size of RT-PCR product(s)

RT-PCR analysis of human ASPM transcripts Exon2-exon3

FN2-3/RN2-3 5'- ctgttaactggacaccactc -3V5¹- 258 bp tgtagtgggctcctaactct -3'

Exonl4-exonl5

FN14-15/RN14-15 5'-agacggccgtgtgttatgtt-3'/5'- 280 bp agcaggtattccaccaaggt -5¹

Exonl-exon28

ASPM1F/ASPM28R S'-tcctgtctctcagccacttc-S'/S¹- 10,315 bp or 5,560 bp* atgccaagcgtatccatcac-3'

F2-3/33-18R1 S'-ctgttaactggacaccactc-S'/S¹- 6,624 bp or 582 bp ctgcattttgtacctgaagga-3 '

F2-3/33-18R2 5'-ctgttaactggacaccactc-375'- 6,686 bp or 644 bp gtgcatctctcgcatccttt-3 '

Exl8R/Exl8F 5'-ataagcacgccaatgcctct-3'/5'- 4,355 bp ccttcagatggctgtgtatc-3 '

Exl8R/AS17F 5'-ataagcacgccaatgcctct-3'/5 '- 277 bp gagttaatgcagcactcgtc-3 '

Exl8F/AS19R S'-ccttcagatggctgtgtatc-S'/S '- 412 bp gcaggaagtatagctctcca-3 '

RT-PCR analysis of mouse Aspm transcripts

ASmIF/ ASm28R 5'-tgctgttgctcagccactt-3V5'- 9,217 bp or 5,574 bp cttccacattatcacgcctc-3'

ASmI 7F/ ASmI 9R 5'-aaggaagttgcggatgctga-3V5'- 4,095 bp or 300 bp acagaaagtgtagctctccatt-3 '

* The second product corresponds to an alternatively spliced variant. 258-bp and 239-bp bands of the expected sizes were detected in all samples, indicating that ASPM is ubiquitously expressed during development (Figure IB).

Because the Drosophila asp homologue is expressed in some adult tissues, human ASPM expression was analyzed in a variety of adult tissues, including breast, lung, pancreas, uterus, colon, thyroid, liver, bladder, kidney, ovary, testis, stomach, lymph node cervix, esophagus, and brain. Human ASPM transcripts were detected in all tissues except adult brain, though the level of expression was much lower than was observed in fetal tissues. The ASPM expression analysis suggested that ASPM functions in non-CNS (non-central nervous system) tissues.

Example 2: ASPM expression is upregulated in cancer Expression data was obtained from over 120 uterine cancers and gene expression was analyzed using high density oligonucleotide microarrays. This analysis identified ASPM as one of several genes highly overexpressed in uterine cancer as compared to normal endometrium. Based on this initial observation, ASPM expression in normal and cancer tissues was analyzed by quantitative real-time RT-PCR. These studies confirmed that ASPM is more highly expressed in cancer of the uterus and ovary as compared to their expression in corresponding normal tissues (Figures 2A and 2B). The analysis of ASPM expression in cancer was expanded by including several other tumor types and obtained similar results for normal and matching tumor tissues of breast, colon, thyroid, testis, lymph, and stomach, as well as for 60 other cancer cell lines. These results are provided in Table 2, below, and in Figure 3 A.

Table 2 Expression of the ASPM gene in NIH-60 cancer cell lines

Sample number Type number Cancer type Cell line Expression

1 Leukemia K562 + 1 Leukemia MOLT4 + 1 Leukemia CCTF CEM + 1 Leukemia RPMI 8226 + 1 Leukemia HL-60 TB + 1 Leukemia SR + 2 CNS SF 268 + 2 CNS SF 295 +

9 2 CNS SF 539 +

10 2 CNS SNB 19 +

11 2 CNS SNB 75 +

12 2 CNS U251 +

13 3 Breast BT549 +

14 3 Breast HS 578 +

15 3 Breast MCF 7 +

16 3 Breast NCIADRRES +

18 3 Breast MDA MB 435 +

19 3 Breast T-47 D +

20 4 Colon Colo 205 +

21 4 Colon HCC 2998 +

22 4 Colon HCT 116 +

23 4 Colon HCT 15 +

24 4 Colon HT29 +

25 4 Colon KM12 +

26 4 Colon SW 620 +

27 5 NSCLC A549 ATCC +

28 5 NSCLC EKVX +

29 5 NSCLC HOP 62 +

30 5 NSCLC HOP 92 +

31 5 NSCLC NCI H 322 M +

32 5 NSCLC NCIH226 +

33 5 NSCLC NCI H23 +

34 5 NSCLC NCIH460 +

35 5 NSCLC NCIH522 +

36 6 Melanoma LOX IMVI +

37 6 Melanoma M14 +

38 6 Melanoma MAL ME +

54 6 Melanoma SK MEL 2 +

55 6 Melanoma SK MEL 5 +

56 6 Melanoma SK MEL 28 +

57 6 Melanoma UACC 62 +

58 6 Melanoma UACC 257 +

39 7 Ovarian IGROV 1 +

40 7 Ovarian OVCAR3 +

41 7 Ovarian OVCAR4 +

42 7 Ovarian OVCAR5 +

43 7 Ovarian OVCAR8 +

44 7 Ovarian SKOV3 +

45 Prostate DU 145 +

46 Prostate PC-3 +

47 Renal 786-0 +

48 Renal A498 +

49 Renal ACHN +

50 Renal CAKI-I +

51 Renal SN12C +

52 Renal TK-10 +

53 Renal UO-31 +

ASPM expression was checked by RT-PCR using primers FN2-3 and RN2-3 developed from exon 2 and exon 3 sequences (see Table 1). Thus, transcription of the human ASPM gene is not restricted to developing brain. The ASPM gene is widely expressed in a variety of adult and embryo tissues and is upregulated in malignancy. Without being tied to one particular theory, it is possible that ASPM is upregulated in malignancy, because its expression correlates with the number of dividing cells in the malignant tissue. Alternatively, ASPM may be required in tumors that are increasing their size just as it is required for increases in brain size in microcephalic individuals.

Example 3: Alternatively spliced variants of human ASPM encode different numbers of IQ domains

The open reading frame (ORF) of ASPM was predicted based on the sequences of multiple small size Expressed Sequence Tags (ESTs) deposited into GenBank, which cover 28 exons. To characterize the human transcript(s), RT-PCR analysis was carried out using primers, ASPMlF and ASPM28R, which are present in the presumed first and last exons of ASPM (Table 1). As seen in Figured 4A and 4B (actin), two predominant PCR products were detected in RNA from fetal tissues. The largest of these RNAs migrated at a size consistent with the predicted full-length mRNA of 10,434 base pairs and was present in all fetal human tissues analyzed. The next most abundant band migrated at approximately 5.6 kb. Additional smaller PCR products were also observed. Similar sized bands were detected in normal as well as in matching tumor tissues (Figure 3A). Expression of mouse ASPM in normal murine tissues is shown in Figure 3B. The predominant PCR products were directly sequenced. The largest human PCR product corresponded to the predicted full-length ORF of 10,434 nucleotides. Further cloning and sequencing of the smaller RTPCR fragments revealed three human mRNAs with open reading frames (ORFs) of 5,678 bp, 4,259 bp and 3,189 bp (GenBank Accession numbers AY971956, AY971957 and AY971955) (Table 3, and Figures 8C-8H). Wild-type ASPM polypeptide and polynucleotide sequences are shown in Figures 8 A and 8B.

Table 3: ASPMcDNA clones and deposited accessions

Identification Species Accessions*

Human splice variant 1 Homo sapiens AY971956

Human splice variant 2 Homo sapiens AY971957

Human splice variant 3 Homo sapiens AY971955

Mouse splice variant 1 Mus musculus AY971958

*GenBaiik accessions correspond to alternatively spliced variants identified in fetal brain and adult tissues. So, in addition to the predicted 3477 amino acid residues protein, the ASPM gene may encode at least three isoforms (Figure 5A and 5B) containing 1892 (variant 1 corresponding to the abundant RT-PCR product), 1389 (variant 2) and 1062 (variant 3) amino acid residues (Figures 8C-8H). The sequence of the wild-type human ASPM polypeptide and the ASPM polynucleotide is provided at Figures 8A and 8B, respectively. The amino acid and nucleic acid sequence of the ASPM variants is provided at Figures 8C-8H. The main difference between these shorter isoforms is primarily in the number of IQ domains (Figures 5A). Specifically, variant 1 lacks exon 18 (carrying 61 IQ domains) leaving this isoform with 14 total IQ domains. Variants 2 and 3 utilize different splice junctions within exon 18 and truncate parts of the IQ array. As a result of this splicing, variant 2 has 41 complete plus one incomplete IQ domains and variant 3 has 27 complete and one incomplete IQ domain. In addition, both variants 2 and 3 lack exons 4-17, which encode two CH (calponin-homology) domains and variant 2 also lacks exon 27, and has a stop codon in the intronic region after exon 26.

To investigate whether alternatively spliced ASPM isoforms are conserved in evolution, RT-PCR analysis was performed using a multiple mouse tissue cDNA panel and a pair of primers that hybridize to the first and last exons of murine Aspm. At least 2 predominant isoforms were detected on ethidium bromide stained gels (Figure 3B). Sequencing of these prominent bands indicated that the largest one was the full-length Aspm of 9,217 bp (containing 67 IQ domains) and the other was an exon 18 deleted isoform of 5,574 bp (15 IQ domains) transcript (accession number AY971958). Both transcripts were abundant in testis and in embryo. The presence of identical spliced ASPM variants lacking exon 18 in human and mouse suggested that this variant encodes the isoform that is required for ASPM function.

Example 4: IQ motifs in the ASPM protein are organized into a higher order repeat structure

More than half of the human ASPM protein consists of repeated calmodulin-binding IQ domains. The IQ array, which spans amino acid positions 1273-3234, is formed by 81 distinct IQ motifs of variable length (Figures 6A-6C). In comparison, the IQ array present in mouse and rat exhibits one large deletion corresponding to a region between IQs 57-70 in human, and several smaller insertions and deletions. In total, 67 IQ repeats are found in mouse Aspm (Figure 6D). The numbers of IQ repeats are slightly higher than previously reported, 74 and 61 repeats for human and mouse, respectively (Bond et al., Nat. Genet. 32, 316-320, 2005; Bond et al., Am. J. Hum. Genet. 73, 1170-1177, 2003) and the difference can be attributed to the very sensitive hidden Markov profile search used in our analysis. The IQ repeat region of ASPM was examined. This analysis showed that the length of individual IQ domains is variable ranging from 14 (IQ 78) to 38 (IQ 76) amino acids (aa). The distribution of variably sized IQ repeats is of random (Figure 6A). The central part of the array between IQ 4-54 displays a striking periodicity with long (27 aa) IQ repeats being dispersed among short (23 aa) IQ repeats. The only exceptions are IQ 43 with 26 instead of 27 aa, and IQs 7-8 with 22 instead of 23aa. The spacing between the long and short units is highly regular. The long IQ 6 repeat is followed by four short units, IQ 11 and IQ 15 by three units. The next 12 long units are in each case followed by two short units. This region forms a highly regular array of 63 aa long superunits, composed of one long 27 aa unit and two short 23 aa units. The N-terminal and C-terminal parts of the IQ repeat region are less regular and contain IQ units of variable lengths. The mouse Aspm protein displays the same periodicity 27-23-23 in the central region of the protein. The N and C terminals of the protein are less organized as is the case of human ASPM (Figure 6D).

The sequence conservation of the long (Figure 6B) and short units (Figure 6C) was also of interest. The data indicated that the consensus sequence is (I,l,v)QX₂(Y,F,w)(k,r)aX_lo(y,f)X₃(k,r)X₃(i,l,v)X for the long unit; (I,l,v)QsX(Y,F,w)(R)Xi₅(i,l,v)X for the short unit. Strongly conserved amino acids are denoted by uppercase letters, less well conserved/minor residues are in lowercase, and "X" ' denotes nonconserved positions. Physico-chemical properties of individual amino acid positions are shown in Figures 6B and 6C.

Finally the pattern of amino acid replacements during primate evolution was analyzed (Figure 6A). Positions identified as conserved in previous analyses were, as expected, mostly conserved among the primates analyzed. The majority of changes in primate ASPM sequences occurred in nonconserved residues. Both conservative and nonconservative substitutions are found in all long, short, and unordered repeats. Similar findings were obtained when the analysis was applied to mouse and rat ASPM sequences (Figure 6D). Interestingly, 66-69 IQ repeats, which are deleted in mice and rats. A higher-order repeat structure of IQ domains in all mammals indicates that this structure is likely to have functional significance. It is worth noting that because the IQ consensus sequence includes two positively charged amino acids, the IQ-containing region that spans 2,000 amino acids is highly positively charged. Among 530 charged residues, 467 are arginine and lysine. This density of basic residues may be required for binding of calmodulin, and/or it may facilitate interactions between ASPM and acidic proteins. Example 5: Identification of a novel ASNP repeat region within ASPM

Analysis of ASPM revealed two novel repeats in the N-terminal part of the protein. The repeats are 32 and 35 amino acids long and localized to positions 316-347 and 366-400, respectively (Figures 5A and 5B). These repeats are highly conserved near the termini, with the central part being less well conserved. Similar repeats were found in all mammalian ASPM homologues. A single repeat was identified in the chicken gene. The interspecies conservation is similar to intraspecies comparisons, i.e. the termini are highly similar, the N- terminus is almost identical, but the central part is variable. The identified repeats are referred to as ASNP (for ASPM N-proximal) repeats. No significant similarity was identified between this motif and other proteins, even when using very sensitive searches (Position-

Specific Iterated Blast (PsiBlast), which was described by Altschul, et al. (Nucleic Acids Res. 25:3389-3402, 1997) and Schaffer, et al. (Nucleic Acids Res. 29: 2994-3005, 2001); and profile hidden Markov model (HMMER) using statistical descriptions of a sequence family's consensus for database searching, as described at the Washington University of St. Louis HMMER website that is maintained by Sean Eddy). Yet, the conservation in the compared species suggests that strong selective pressure preserves the repeats, indicating their functional importance for ASPM function.

Example 6: ASPM is found at the spindle poles during mitosis in human culture cells The role of Drosophila Asp in nucleating microtubules at centrosomes is consistent with its localization at the spindle poles during mitotic periods (Saunders et al., J. Cell. Biol. 137, 881-890, 1997). Polyclonal antibodies against three epitopes of human ASPM were generated. The anti-ASPM antibodies for two different epitopes, VTRK and QSPE, were used for indirect immunostaining of human HT1080 cultured cells. The VTRK antibody showed strong and similar typical spindle pole staining pattern during prometaphase, metaphase and anaphase (Figures 7A-I). A characterization of the VTRK antibody is shown at Figure 7 J, 7K, and 7L. A similar staining pattern was observed with the QSPE antibody during mitotic periods. The results of the mitotic subcellular localization and the highly conserved structural homology between Drosophila Asp and human ASPM suggested that human ASPM may be involved in a conserved spindle function.

As described herein, human ASPM is a mitotic spindle protein that is likely a functional orthologue of the Drosophila asp protein Bond et al., Nat. Genet. 32, 316-320 (6). This conclusion is supported by a recent analysis of proteins of the purified human mitotic spindles Sauer et al., (2005) Proteome analysis of the human mitotic spindle. MoI. Cell. Protβomics 4, 35-43 (23) which provides evidence that microcephaly arises from deficient neurogenic mitosis. Without being tied to one particular theory, it is possible that as a component of the mitotic spindle, ASPM controls the proliferative symmetry of progenitors required for the expansion of cerebral cortical size Chenn et al., Cereb. Cortex 13, 599-606 (24). Alternatively, the presence of a nonfunctional ASPM might reduce chromosome segregation fidelity, resulting in a high incidence of chromosome aneuploidy, and reducing the ability of fetal stem cells to produce neurons.

Bond and co-authors identified two additional MCPH genes, MCPH3 and MCPH6 Bond et al., Nat. Genet. 37, 353-355 (4). It is notable that both of these genes encode spindle pole proteins. These findings, when taken together with the present studies, indicate a key role for the centrosome in the regulation of neurons generated by neural precursor cells. Identification of molecular partners of human ASPM would elucidate the molecular mechanisms controlling neuron generation. Such molecular partners might include the kinase Polo, which phosphorylates Drosophila Asp and co.-purifies with γ-tubulin, which is present in centrosomes Avides et al., Science 283, 1733-1735, Avides et al., Nat. Cell. Biol. 4, 421- 424, Saunders et al., The Drosophila gene abnormal spindle encodes a novel microtubule- associated protein that associates with the polar regions of the mitotic spindle. J. Cell. Biol. 137, 881-890 (18, 19, 21).

As for many other brain-expressed genes Andreadis, Biochim. Biophys. Ada. 1739, 91-103 (25), ASPM encodes several alternatively spliced mRNAs. The main ASPM isoform is a 3477 amino acid residue protein that contains eighty-one IQ motifs. Most of these are organized into a Higher-Order trimer Repeat (HOR) containing two units each comprising 23 and 27 amino acid residues. The HOR structure of the IQ array is conserved in primates and mouse ASPM proteins, suggesting that such a structure is essential for a protein function. Interestingly, five IQ repeats of myosin V are also organized into a higher-order structure, but the IQ domain is much shorter and forms a 25-23-25-23-25 amino acid residues array. Myosin V is an unconventional myosin that transports cellular cargos such as vesicles, melanosomes, or mRNA on actin filaments. The IQ periodicity seems to be necessary for efficient interaction between myosin V IQ domains and actin half-repeats, which are 36-nm long Sakamoto et al., J. Boil. Chem. 278, 29201-29207 (26). Analogously, the ASPM IQ periodicity may provide the exact spacing required for interactions with polymeric periodical proteins such as actin. The major alternatively spliced form contains an in frame deletion of the entire exon 18 sequence and encodes 1892 amino acid residues with the predicted protein harboring fourteen flanking IQ domains that do not exhibit any periodicity. Similarly, two predominant transcripts were also present in mouse. In addition, alternatively spliced variants lacking both CH domains and more than a half of mostly periodical IQ domains were also detected, suggesting that ASPM may encode several proteins with different functions.

The presence of a long array of putative calmodulin-binding IQ domains is a unique feature of ASPM Bahler et al., (2002) FEBS Lett. 513, 107-113 (13). It has the highest number of the domains as compared to more than hundred IQ-containing proteins identified so far. Even if some of them are not functional, potentially ASPM could bind more calmodulin molecules than any other known IQ-containing protein. Calmodulin appears to regulate a protein function by modulating its ability to bind to other targets. This can occur by 2 mechanisms: i) by altering the conformation of the protein as well as ii) by influencing its subcellular location. Both the copy number and HOR structure of the calmodulin-binding domains in ASPM could greatly affect both mechanisms. The presence of multiple domains (IQ, CH and ASNP identified in this work) within ASPM also suggest its additional function such as a scaffolding protein that assembles multiprotein complexes.

If, similar to the Drosophila orthologue, human ASPM is involved in nucleating microtubules at centrosomes Saunders et al., The Drosophila gene abnormal spindle encodes a novel microtubule-associated protein that associates with the polar regions of the mitotic spindle. J. Cell. Biol. 137, 881-890, Avides et al., Science 283, 1733-1735, Avides et al., Nat. Cell. Biol. 4, 421-424, (20, 18, 19), a specific role of IQ repeats may be accumulation of Ca⁺²/calmodulins at the central region of the protein. Potentially one molecule of ASPM protein can bind through calmodulin several hundred calcium ions. Release of the bind Ca⁺² may signal microtubule polymerization.

The results reported herein indicate that ASPM is widely expressed in adult tissues and is upregulated in cancer cells. The role of ASPM during mitosis of non-CNS (non-central nervous system) cells or its transcription may simply reflect proliferation of a tissue. Without being tied to one particular theory, given the localization of the ASPM gene product(s) at the mitotic spindle it is likely that some mutations (or polymorphic variants) that do not compromise a specific function of ASPM in neural progenitors may compromise the fidelity of cell division and cause chromosome instability in Adult tissues. Intriguingly, defects of the centrosome has been found in numerous forms of cancers Wang et al., DNA Cell. Biol. 23, 475-489, Marumoto T. et al., Nat. Rev. Cancer. 5, 42-50 (27, 28). Therefore, further studies of ASPM polymorphism and function of the identified isofoπns will clarify molecular mechanisms of microcephaly, and determine whether specific mutations in the human ASPM nucleic acid sequence indicate a predisposition to cancer.

The experiments reported herein were carried out using the following materials and methods.

Mouse embryo in situ hybridization

Non-radioactive in situ hybridization was performed using a digoxigenin (DIG)- labelled cRNA probe. The antisense probe was generated from a mouse EST clone (Genbank accession: AW558815) using standard methods, and frozen sections hybridized and visualized using methods described previously (Berger et al., Jour. Comp. Neurol. 433, 101- 114, 2001).

Analysis of fetal and adult tissues by RT-PCR Total RNAs from fetal (brain, bladder, colon, testis, liver, heart, liver, skeletal muscle, skin, spleen, stomach), adult (brain, breast, lung, pancreas, uterus, colon, thyroid, liver, bladder, kidney, ovary, testis, stomach, lymph node, cervix, esophagus) and matching cancer human tissues, mouse (brain, testis, liver, heart) tissues (Ambion) were used for screening ASPM expression with the primers described in Table 1. cDNA was made from 1 μg of total RNA using a commercially available kit for reverse transcriptase polymerase chain reaction (RT-PCR), SUPERSCRIPT l^sτ STRAND SYSTEM kit (Invitrogen, Carlsbad, CA), and priming with oligo dT per their standard protocol. Human β-actin primers (BD Biosciences Clontech, Palo Alto, CA) were used as positive controls for both human and mouse RT-PCR. NCI-60 cancer cell lines were from the National Cancer Institute, NIH. RT-PCR was performed using 1 μl of cDNA in a 50 ul reaction volume. Standard reaction conditions were: 94°C 5m, (94°C 1 minute, 55°C 1 minute, 72°C 1 minute x 35 cycles), 72°C 7 minutes, 4°C hold. Sequencing of the RT-PCR products confirmed their identity to the ASPM transcripts.

Analysis of cancer tissues by real-time PCR RNA levels of ASPM were assessed in clinical specimens. The relative expression of

ASPM was assayed by quantitative PCR using a commercially available assay system, FAM- labeled TaqMan® Gene Expression Assays, from Applied Biosystems, (Foster City, CA). Detectably labeled β-actin, VIC-labeled β-actin (4326315E), was used as a reference. Samples were run on the ABI Prism® 7700 Sequence Detection System according to manufacturer's suggested Protocols in separate tubes. The relative quantitation, using the comparative Cx method, was calculated for each sample.

Sequencing of ASPM spliced forms

The full-length ASPM transcripts (10,434 bp for human and 9,846 bp for mouse) were sequenced after cloning of RT-PCR products into a TA cloning vector (Invitrogen, Carlsbad, CA). The RT-PCR products corresponding to human ASPM alternatively spliced variants of 5,679 bp, 4,259 bp and 3,189 bp and mouse spliced variant of 5,574 bp were also sequenced. Sequence forward and reverse reactions were run on a PE- Applied Biosystem 3100

Automated Capillary DNA Sequencer with a set of the designed primers. Comparative analyses were performed with the wild-type cDNA sequences (Genbank accession number gi:24211028 and gi:36031058 for human and mouse) using a publically available sequence analysis software, GCG DNA Analysis Wisconsin Package and NCBI BLAST. All sequenced clones were named and numbered according to the clone/accession identifier (Table 3).

Sequence analysis and identification of IQ repeats

Sequences were aligned by Dialign2.1 Morgenstern, B. (1999) Bioinformatics 15, 211-218 (30); http://bibiserv.techfak.uni-bielefeld.de/dialign/. Protein alignments were visualized using publically available software, GeneDoc (Nicholas et al., EMBNEW.NEWS 4, 14); http://www.psc.edu/biomed/ genedoc and WAVis [(Zika et al., Nucl. Acids Res. 32:W48-49, 2004); http://wavis.img.cas.cz. Conserved domains in the ASPM protein were detected by Rpsblast with default parameters (Marchler-Bauer et al., Nucleic Acids Res. 32, W48-49); http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi]. The human ASPM protein was initially screened for IQ repeats by Radar fhttp://www.ebi.ac.uk/Radar/). The detected IQ repeats were used to build a hidden Markov profile using publically available software, HMMER v 2.2 Eddy, Bioinformatics 14, 755-763 (34); http://hmmer.wustl.edu/. In the next step, the obtained profile was used to search for the remaining IQ repeats. The IQ sequences were manually edited in order to obtain exact boundaries of all IQ units. This complete set was used to build a new hidden Markov profile, which subsequently served for detection of IQ repeats in mouse Aspm. Other ASPM repeats, including newly detected N-terminal repeats were identified by Radar. Peptide specific antibodies and Western blot analysis

Three synthetic peptides representing amino acids 422-441

(QSPEDWRKSEVSPRIPE), 497-520 (VTKRKATCTRENOTEINKPKAKR), and 3443- 3463 (SRLKPDWVLRRDNMEEITNPL) from the ASPM sequence were conjugated to Keyhole Limpet Hemocyanin and used as immunogenes as previously described (Goldsmith , et al., Biochemistry 27, 7085-7090, 1988). The resulting antisera were affinity purified over columns of peptide conjugated to Affigel 15 (Bio-Rad, Richmond, CA), and concentrated in stirred cells with YM30 membranes (Millipore, Billerica, MA). The concentrates were then subjected to gel filtration chromatography using 2.6 x 60 cm SUPERDEX 200 columns (GE Healthcare, Piscataway, NJ) and the monomeric IgG fractions were pooled and concentrated. The protein concentrations were then determined using a commercially available kit, the Bradford assay (Bio-Rad, Richmond, CA). These peptides were identical to both human and mouse ASPM proteins. The antibodies specificity for these peptides was first confirmed with recombinant ASPM fragments. Plasmids for ASPM expression in E.coli cells were constructed by insertion of a 1353 bp fragment containing QSPE and VTKR epitopes, and a 705 bp fragment containing SRLK epitope. The fragments were PCR amplified from the full-size ASPM cDNA (positions 346-1695 and 9730-10434) and cloned into BatήBI site of the pMAL-p2X expression vector (BioLabs Inc.). To analyze ASPM protein in mammalian cells, human (HTl 080) and mouse (3T3) cells were mixed with SDS sample buffer containing a protease inhibitor cocktail (Sigma), homogenized using a 27 gauge needle and resolved in a 3.4% of 29:1 acrylamide/bis-acrylamide gel. Following electrophoresis, the proteins were transferred to PVDF membranes (Millipore) for 40 minutes at 15V in transfer buffer (50 mM Tris, 380 mM glycine, 0.1% SDS and 20% methanol) using a semi-dry method. All subsequent steps were carried out in phosphate buffered saline (PBS) containing 0.05% Tweet' 20 (TPBS). After thirty minutes blocking with 10% TPBS nonfat milk, the membranes were exposed for 12 hours to 1/2,000 diluted anti-QSPE or SRLK antibodies. The PVDF membrane was washed three times with TPBS, then incubated with anti-rabbit IgG conjugated with horse radish peroxidase (HRP) diluted 1/5000 for 30 minutes, and then washed as in the previous step. The membranes were incubated for 1 minute with commercially available chemiluminescent detection reagents, ECL plus reagents (Amersham) and exposed to Hperfilm ECL (Amersham). Immunoblotting of cellular proteins from human HTl 080 and mouse 3T3 cells revealed the predicted protein isoforms corresponding to two main alternatively spliced variants. These bands were not detected in a lymphoblast cell line derived from a microcephalic individual with a frameshift mutation in ASPM (Bond et al., Am. J. Hum. Genet. 73, 1170-1177) indicating that the antibodies are highly specific (Figure 7J, K₅ L).

Indirect immunofluorescence staining with anti-ASPM antibodies For detection of the endogenous ASPM protein, two different fixation methods were employed with HT1080 cells grown on poly-L-lysine coating coverslips. 1) Cultured cells were first incubated for 15 minutes with 2% paraformaldehyde. Samples were washed twice and then treated for 5 minutes with 0.5% Triton X-100 (Sigma) and for 5 minutes with 0. IM Glycine (Sigma). 2) Cultured cells were treated for 30 minutes with 100% methanol (Mallinckrodt Baker). Fixed samples were incubated for 1 hour at 37°C with anti-ASPM antibodies (1 :200). After washing three times with PBS for 5 minutes each, samples were incubated for 1 hour at 37°C with a commercially available fluorescent dye, Alexa Fluor 594 coupled to goat anti-rabbit IgG (Molecular Probes). After washing three times with PBS for 5 minutes each, samples were stained with lμg/ml of DAPI and rinsed again with PBS. Samples were finally mounted using Vectashield mounting medium (Vector Lab). Images were captured using a Zeiss microscope (Axiophoto) equipped with a cooled-CCD camera (Cool SNAP HQ, Photometries Ltd) and analyzed by IPLab software (Signal Analytics).

Other Embodiments A number of embodiments of the invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention.

Claims

What is claimed:

1. A method of diagnosing a subject as having, or having a propensity to develop, a neoplasia, the method comprising determining the level of expression of an ASPM nucleic acid molecule in a subject sample, wherein an increased level of expression relative to a reference, indicates that the subject has or has a propensity to develop a neoplasia.

2. A method of diagnosing a subject as having, or having a propensity to develop, a neoplasia, the method comprising determining the level of expression of an ASPM polypeptide in a subject sample, wherein an increased level of expression relative to the level of expression in a reference, indicates that the subject has or has a propensity to develop a neoplasia.

3. The method of claim 2, wherein the method comprises determining the level of expression of the ASPM polypeptide.

4. The method of claim 3, wherein the level of expression is determined in an immunological assay.

5. A method of diagnosing a subject as having, or having a propensity to develop, a neoplasia, the method comprising determining the level of biological activity of an ASPM polypeptide in a subject sample, wherein an alteration in the level of biological activity relative to the biological activity in a reference, indicates that the subject has or has a propensity to develop a neoplasia.

6. A method of monitoring a subject diagnosed as having a neoplasia, the method comprising determining the level of activity of an ASPM polypeptide in a subject sample, wherein an alteration in the level of expression relative to the level of expression in a reference indicates the severity of neoplasia in the subject.

7. The method of claim 6, wherein the subject is being treated for a neoplasia.

8. The method of claim 7, wherein the neoplasia is breast, prostate, lung, testis, ovary, or uterine neoplasia.

9. The method of claim 6, wherein the alteration is an increase, and the increase indicates an increased severity of neoplasia.

10. The method of any one of claims 1-6, wherein the reference is a control subject sample.

11. The method of claim 6, wherein the reference is a subject sample obtained at an earlier time point.

12. The method of any one of claims 1-6, wherein the subject sample is a biological sample.

13. The method of any one of claims 1-7, wherein the method is used to diagnose a subject as having neoplasia.

14. The method of any one of claims 1-7, wherein the method is used to determine the treatment regimen for a subject having neoplasia.

15. The method of any one of claims 1-7, wherein the method is used to monitor the condition of a subject being treated for neoplasia.

16. The method of any one of claims 1 -7, wherein the method is used to determine the prognosis of a subject having neoplasia.

17. The method of claim 16, wherein a poor prognosis determines an aggressive treatment regimen for the subject.

18. A method for identifying a subject as having or having a propensity to develop a neoplasia, the method comprising detecting an alteration in the sequence of an ASPM nucleic acid molecule relative to the sequence or expression of a reference molecule.

19. The method of claim 18, wherein the alteration is detected using a hybridization reaction.

20. An ASPM antibody that specifically binds to an ASPM protein or fragment thereof.

21. The antibody of claim 20, wherein the antibody binds to a VTRK or QSPE epitope of an ASPM polypeptide.

22. A polypeptide comprising an isolated ASPM protein variant, or fragment thereof, having substantial identity to ASPM variant 1, 2, or 3, wherein the variant is upregulated in a neoplastic cell.

23. The polypeptide of claim 22, wherein the ASPM protein variant is at least 85% identical to ASPM variant 1, 2, or 3.

24. The polypeptide of claim 22, wherein the ASPM protein variant comprises at least an IQ domain and is capable of binding calmodulin.

25. The polypeptide of claim 22, wherein the fragment consists of an ASPM protein variant selected from the group consisting of ASPM variant 1, 2, and 3.

26. The polypeptide of any one of claims 22-25, wherein the polypeptide is a fusion protein.

27. The polypeptide of any one of claims 22-25, wherein the polypeptide is linked to a detectable amino acid sequence.

28. The polypeptide of any one of claims 22-25, wherein the polypeptide is linked to an affinity tag.

29. An isolated ASPM nucleic acid molecule, wherein the nucleic acid molecule encodes a polypeptide of any one of claims 22-28.

30. A vector comprising the nucleic acid molecule of claim 29.

31. An isolated ASPM inhibitory nucleic acid molecule, wherein the inhibitory nucleic acid molecule specifically binds at least a fragment of a nucleic acid molecule encoding an

ASPM protein.

32. A vector comprising a nucleic acid molecule encoding the nucleic acid molecule of claim 31.

32. The vector of claim 30 or 32, wherein the vector is an expression vector.

33. The vector of claim 30 or 32, wherein the nucleic acid molecule is positioned for expression.

34. The vector of claim 30 or 32, wherein the nucleic acid molecule is operably linked to a promoter.

35. The vector of claim 30 or 32, wherein the promoter is suitable for expression in a mammalian cell.

36. A host cell comprising a nucleic acid molecule of any one of claims 29-35.

37. The host cell of claim 36, wherein the cell expresses an ASPM protein variant.

38. The host cell of claim 36, wherein the cell is in vitro.

39. The host cell of claim 36, wherein the cell is in vivo.

40. The host cell of claim 36, wherein the cell is a mammalian cell.

41. The host cell of claim 36, wherein the cell is a human cell.

42. A double-stranded RNA corresponding to at least a portion of an ASPM nucleic acid molecule that encodes an ASPM protein, wherein the double-stranded RNA is capable of altering the level of protein encoded by the ASPM nucleic acid molecule.

43. The RNA of claim 42, wherein the RNA is an siRNA.

44. An antisense nucleic acid molecule, wherein the antisense nucleic acid molecule is complementary to at least six nucleotides of an ASPM nucleic acid molecule that encodes an ASPM protein, and wherein the antisense is capable of altering expression from the nucleic acid molecule to which it is complementary.

45. A primer capable of binding to an ASPM nucleic acid molecule encoding an ASPM protein variant that is upregulated in a neoplastic tissue.

46. A collection of primers capable of binding to and amplifying an ASPM nucleic acid molecule, wherein at least one of the primers in the collection is the primer of claim 22.

47. A pharmaceutical composition comprising an effective amount of an ASPM protein, variant, or fragment thereof, in a pharmaceutically acceptable excipient, wherein the fragment is capable of modulating cell proliferation or chromosome stability.

48. A pharmaceutical composition comprising an effective amount of a nucleic acid molecule of any one of claims 29-44 in a pharmaceutically acceptable excipient, wherein the fragment is capable of modulating cell proliferation or chromosomal stability.

49. A pharmaceutical composition comprising an effective amount of a vector comprising a nucleic acid molecule encoding an ASPM protein of any one of claims 29-44 in a pharmaceutically acceptable excipient, wherein expression of the ASPM protein in the cell is capable of modulating cell proliferation or chromosomal stability.

50. The pharmaceutical composition of claim 49, wherein the ASPM nucleic acid molecule is positioned for expression in a mammalian cell.

51. An ASPM biomarker purified on a biochip.

52. A microarray comprising at least two nucleic acid molecules, or fragments thereof, fixed to a solid support, wherein at least one of the nucleic acid molecules is an ASPM nucleic acid molecule.

53. A microarray comprising at least two polypeptides, or fragments thereof, bound to a solid support, wherein at least one of the polypeptides on the support is an ASPM polypeptide.

54. A diagnostic kit for the diagnosis of a neoplasia in a subject comprising an ASPM nucleic acid molecule, or fragment thereof, and written instructions for use of the kit for detection of a neoplasia.

55. A diagnostic kit for the diagnosis of a neoplasia in a subject comprising an antibody that specifically binds an ASPM polypeptide, or fragment thereof, and written instructions for use of the kit for detection of a neoplasia.

56. A kit identifying a subject as having or having a propensity to develop a neoplasia, comprising an adsorbent, wherein the adsorbent retains an ASPM biomarker, and written instructions for use of the kit for detection of a neoplasia.

57. A kit comprising a first capture reagent that specifically binds an ASPM biomarker, and written instructions for use of the kit for detection of a neoplasia.

58. A method for detecting neoplasia in a subject sample, the method comprising

(a) contacting a subject sample with a capture reagent affixed to a substrate; and

(b) capturing an ASPM polypeptide or nucleic acid molecule with the capture reagent.

59. The method of claim 58, wherein the subject sample comprises an ASPM protein or fragment thereof.

60. The method of claim 58, wherein the proteins are fractionated prior to contacting the capture reagent.

61. A method of altering the expression of an ASPM nucleic acid molecule in a cell, the method comprising contacting the cell with an effective amount of a compound capable of altering the expression of the ASPM nucleic acid molecule .

62. The method of claim 61 , wherein the compound is an ASPM antisense nucleic acid molecule, a small interfering RNA (siRNA), or a double stranded RNA (dsRNA) that inhibits the expression of an ASPM nucleic acid molecule.

63. A method of altering ASPM protein expression in a cell, the method comprising contacting the cell with a compound capable of altering the expression of an ASPM polypeptide.

64. The method of claim 61 or 63, wherein the cell is a human cell.

65. The method of claim 61 or 63, wherein the cell is a neoplastic cell.

66. The method of claim 61 or 63, wherein the cell is in vitro.

61. The method of claim 61 or 63, wherein the cell is in vivo.

68. A method of treating or preventing a neoplasia, the method comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition that alters expression of an ASPM polypeptide.

69. A method of identifying a compound that inhibits a neoplasia, the method comprising contacting a cell that expresses an ASPM nucleic acid molecule with a candidate compound, and comparing the level of expression of the nucleic acid molecule in the cell contacted by the candidate compound with the level of expression in a control cell not contacted by the candidate compound, wherein an alteration in expression of the ASPM nucleic acid molecule identifies the candidate compound as a compound that inhibits a neoplasia.

70. The method of claim 69, wherein the alteration in expression is a decrease in transcription.

71. The method of claim 69, wherein the alteration in expression is a decrease in translation.

72. A method of identifying a compound that inhibits a neoplasia, the method comprising contacting a cell that expresses an ASPM polypeptide with a candidate compound, and comparing the level of expression of the polypeptide in the cell contacted by the candidate compound with the level of polypeptide expression in a control cell not contacted by the candidate compound, wherein an alteration in the expression of the ASPM polypeptide identifies the candidate compound as a compound that inhibits a neoplasia.

73. A method of identifying a compound that inhibits a neoplasia, the method comprising contacting a cell that expresses an ASPM polypeptide with a candidate compound, and comparing the biological activity of the polypeptide in the cell contacted by the candidate compound with the level of biological activity in a control cell not contacted by the candidate compound, wherein an alteration in the biological activity of the ASPM polypeptide identifies the candidate compound as a candidate compound that inhibits a neoplasia.

74. The method of any one of claims 68-73, wherein the cell is in vitro.

75. The method of any one of claims 68-73, wherein the cell is in vivo.

76. The method of any one of claims 68-73, wherein the cell is a human cell.

77. The method of any one of claims 68-73, wherein the cell is a neoplastic cell.

78. The method of any one of claims 68-73, wherein the alteration in expression is assayed using an immunological assay, an enzymatic assay, or a radioimmunoassay.

79. A method of identifying a candidate compound that inhibits a neoplasia, the method comprising a) contacting a cell containing an ASPM nucleic acid molecule present in an expression vector that includes a reporter construct; b) detecting the level of reporter gene expression in the cell contacted with the candidate compound with a control cell not contacted with the candidate compound, wherein an alteration in the level of the reporter gene expression identifies the candidate compound as a candidate compound that inhibits a neoplasia.