WO2010068850A1 - Methods of diagnosing and treating cancer - Google Patents

Methods of diagnosing and treating cancer Download PDF

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WO2010068850A1
WO2010068850A1 PCT/US2009/067647 US2009067647W WO2010068850A1 WO 2010068850 A1 WO2010068850 A1 WO 2010068850A1 US 2009067647 W US2009067647 W US 2009067647W WO 2010068850 A1 WO2010068850 A1 WO 2010068850A1
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method
ptprd
cancer
substitution
subject
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PCT/US2009/067647
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French (fr)
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Todd A. Waldman
David Solomon
Jung-Sik Kim
Yardena Samuels
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Georgetown University
The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Publication of WO2010068850A1 publication Critical patent/WO2010068850A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00Oligonucleotides characterized by their use
    • C12Q2600/156Polymorphic or mutational markers

Abstract

Provided are methods of determining whether a subject has or is at risk for developing cancer including determining whether the subject has one or more mutations in protein tyrosine phosphatase receptor type D (PTPRD). One or more mutations in PTPRD indicates the subject has or is at risk for developing cancer. Also provided are methods of treating or preventing cancer in a subject including administering a composition comprising a pharmaceutically acceptable excipient and PTPRD or a nucleic acid encoding PTPRD to the subject.

Description

Methods of Diagnosing and Treating Cancer

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Serial No. 61/122,164 filed December 12, 2008, which is incorporated by reference herein in its entirety.

BACKGROUND

Cancer is a disease that results from the disruption of signaling pathways that regulate cellular proliferation, differentiation, and programmed cell death. This disruption has a genetic basis. Most current research supports the notion that the majority of cancer-causing genes contribute to neoplasia at low frequency and in a limited tumor spectrum. Thus, the discovery of new oncogenes and tumor suppressor genes associated with tumorigenesis or the risk of tumorigenesis remains a major goal of modern cancer research, since such genes and the pathways they control are potential targets for anticancer drug development.

SUMMARY Provided are methods of determining whether a subject has or is at risk for developing cancer including, for example, melanoma, Ewing sarcoma, primitive neuroectodermal tumors and glioblastoma. Specifically, the method comprises obtaining a biological sample (e.g., a neoplastic sample or a non-neoplastic sample) from the subject and determining in the sample whether protein tyrosine phosphatase receptor type D (PTPRD) comprises one or more mutations. The mutations include, for example, mutations in the nucleic acid encoding PTPRD selected from the group consisting of G182A, G1891A, G1337A, C1889T, G1882A, G2507A, G2510A, A2626T, G3007A, G3460A, G3886A, G3835T, C3836T, C4429T, T3486C, C1421T, C1518T, G3957T, C1421T, C1518T, G3957T, G324A, G1235A, G1479A, G3266A, G3518A, G3884A, G4474A, G4475A, G4835A, C5210T, C5211T, C5533T, and

C5534T. Optionally, the mutations include, for example, mutations in the amino acid sequence encoding PTPRD selected from the group consisting of G61E, E631K, G446E, P630L, E628K, M836I, D837N, K876X, G1003R, Vl 1541, G1296R, P1279S, P1279L, P1279F, H1477Y, R427Stop, P459L, I1115T, G1272V, G61E, E365K, G446E, E1042K, V1126M, D1248N, W1444Stop, V1565I, P1690F, and R1798Stop. One or more mutations in PTPRD indicates the subject has or is at risk for developing cancer. Further provided is a method of detecting whether a subject has or is at risk for developing cancer by determining whether the subject has an inherited mutation in

PTPRD.

Also provided are methods of treating or preventing cancer. Specifically, the methods comprise administering a composition comprising a pharmaceutically acceptable excipient and PTPRD or a nucleic acid sequence encoding PTPRD to a subject. Optionally, the methods further comprise administering to the subject a therapeutic agent or radiation therapy or a combination thereof.

DESCRIPTION OF DRAWINGS

Figure 1 shows that protein tyrosine phosphatase receptor D (PTPRD) is deleted at high frequency in glioblastoma multiforme (GBM). Figure IA is a table showing the most frequently deleted genes in 58 GBM tumor samples as determined by Affymetrix 250K SNP microarray analysis. Figure IB shows copy number analysis of SNP microarray data that demonstrates focal (<10 Mb) deletions of chromosome 9p23-24.1 in 8 of 58 GBM samples (four shown) but not in normal human astrocytes (NHAs). x, primary xenograft. Figure 1C shows copy number analysis of SNP microarray data demonstrates large-scale (>10 Mb) chromosomal loss of the PTPRD locus in 19 of 58 GBM samples (five shown), p, primary tumor; c, primary culture.

Figure 2 shows the identification of somatic and inherited mutations of PTPRD in GBM. Figure 2A is a table showing one nonsense and three missense mutations identified in GBM samples. Λ, Genomic position is based on the hgl8 genome assembly. #, Transcript ENST00000381196 was used for annotation of the nucleotide and amino acid changes. *, Assignment of functional domains was based on UniProtKB/Swiss-Prot P23468-1. FN III, fibronectin type-III domain. Figure 2B shows sequence traces depicting an inherited heterozygous mutation of PTPRD in constitutional DNA (blood) and somatic loss of the wild-type allele in GBM primary tumor pi 118. Figure 2C shows sequence traces depicting somatic nonsense and missense mutations of PTPRD in constitutional DNA (blood) and somatic loss of the wild-type allele in GBM primary tumors pi 151 and p898.

Figure 3 shows sequence traces depicting somatic single-nucleotide point mutations of PTPRD in constitutional DNA (blood) and somatic loss of wild-type allele in malignant melanoma primary tumors 34T, 21T, and 86T.

Figure 4 shows sequence traces depicting somatic dinucleotide mutations of PTPRD in constitutional (blood) and somatic loss of wild-type allele in malignant melanoma primary tumors 76T and 6T.

Figure 5 is a schematic of the PTPRD protein and the location of all mutations reported to date in human cancer. S, signal peptide; Ig, immunoglobulin-like C2-type domain; FN, fibronectin type-Ill domain; broken line, cleavage site; PTPc, protein tyrosine phosphatase catalytic domain.

Figure 6 shows that the expression of PTPRD in GBM and melanoma cells harboring deletions and mutations causes growth suppression and apoptosis. Figure 6A is a Western blot for PTPRD demonstrating reconstitution of PTPRD expression in 8MGB A and H4 GBM cells infected with lenti-PTPRD. The C- 18 antibody recognizes a C-terminal epitope present on both the full-length PTPRD proprotein (-175 kDa) and one of the two mature cleavage products (~75 kDa). Figure 6B shows flow cytometry of H4 GBM cells at 48 hours post-infection revealing that lenti-PTPRD causes a 32% reduction in BrdU incorporation and a 2.5-fold increase in sub-Gl cells. Figure 6C shows phase contrast microscopy of cells ten days postinfection that demonstrates reconstitution of PTPRD expression leads to cell death in 16T and 86T melanoma cells harboring homozygous missense mutations of PTPRD (Table 1). Figure 6D shows that Lenti-PTPRD causes a time dependent increase in apoptosis in 16T and 86T cells. Hoechst-stained nuclei of cells undergoing apoptosis after infection with lenti-PTPRD are depicted in the upper panel, and quantification of apoptotic cells is depicted in the lower panel.

Figure 7 shows that tumor-derived mutations compromise the growth suppressive function of PTPRD in GBM and melanoma cells. Figure 7 A is a Western blot analysis for PTPRD protein that demonstrates equivalent expression of wild-type and mutant proteins in infected H4 GBM cells. Figure 7B is a bar graph that demonstrates that infection of H4 cells with wild-type PTPRD led to growth suppression, as measured by BrdU incorporation using a 1 hr. pulse 48 hours after infection. In contrast, ectopic expression of the PTPRD cDNA harboring tumor- derived mutations led to less potent growth suppression. (p<0.05 for each of the mutants, except D1248N with/?=0.06). Figure 7C is a bar graph demonstrating that infection of 16T melanoma cells with wild-type PTPRD led to apoptosis of approximately 75% of the infected cells at 10 days post-infection, whereas the mutant PTPRD lentiviruses led to a substantially reduced fraction of cells that had undergone programmed cell death.

DETAILED DESCRIPTION Constitutive activation of tyrosine phosphorylation signaling pathways is one biochemical hallmark of cancer. This is most well known to occur via activation of tyrosine kinase receptors, such as amplification HER2/Neu in breast cancer and mutation of EGFR in lung cancer. Though inactivating mutations of individual protein tyrosine phosphatases (PTPs) have recently been reported in human colon cancer, at present there is no single tyrosine phosphatase thought to play a generally important role as a tumor suppressor gene in multiple tumor types.

PTPRD is one of 21 known human receptor-type PTPs, a group of genes which are thought to be important in cancer development and progression.

As described in the examples below, frequent deletion and mutation of PTPRD was identified in glioblastoma multiforme and malignant melanoma and it was demonstrated that these mutations are inactivating. These data provide the first evidence that PTPRD is a tumor suppressor gene and show that inactivation of PTPRD contributes to the pathogenesis of a wide range of common human cancers. Provided herein are methods of determining whether a subject has or is at risk for developing cancer, for example, melanoma or glioblastoma. Optionally, the type of cancer a subject has or is at risk for can be selected from a list consisting of brain cancer, lung cancer, breast cancer, prostate cancer, colon cancer, stomach cancer, pancreatic cancer, bone cancer, soft tissue cancer, and skin cancer. Optionally, the skin cancer is melanoma. Optionally, the type of cancer is brain cancer. Optionally, the brain cancer is a glioblastoma. Optionally, the type of cancer is Ewing's Sarcoma.

Optionally, the cancer is a primitive neuroectodermal tumor. As used herein, a subject at risk of developing a disease or disorder can be genetically predisposed to the disease or disorder, e.g., have a family history or have a mutation in a gene that causes the disease or disorder, or show early signs or symptoms of the disease or disorder. A subject currently with a disease or disorder has one or more than one symptom of the disease or disorder and may have been diagnosed with the disease or disorder.

Specifically, the method comprises obtaining a biological sample from the subject and determining in the sample whether protein tyrosine phosphatase receptor type D (PTPRD) comprises one or more mutations. One or more mutations in PTPRD indicates the subject has or is at risk for developing cancer, for example, glioblastoma, Ewing's sarcoma, primitive neuroectodermal tumor or melanoma. Optionally, the mutation is a substitution or a deletion. Optionally, the deletion results in a premature stop. For example, the nucleotide A2197 is deleted resulting in a premature stop at amino acid 735. Optionally, the determining step includes detecting one or more mutations in a nucleic acid encoding PTPRD. Optionally, the determining step includes detecting one or more mutations in the amino acid sequence of PTPRD. As used herein, PTPRD refers to the protein tyrosine phosphatase receptor D, which acts to remove phosphate groups from tyrosine residues in proteins involved in signaling pathways that regulate cellular proliferation, differentiation, and programmed cell death. The sequences of PTPRD are disclosed on GenBank and these sequences are herein incorporated by reference in their entireties as are individual subsequences or fragments contained therein. For example, the nucleic acid sequence of human PTPRD can be found at GenBank Accession No. NM 002839. The amino acid sequence of PTPRD can be found at GenBank Accession No. NP_002830.

As discussed above, the determining step includes detecting one or more mutations in the amino acid sequence of PTPRD. The mutation can, for example, be a substitution selected from the group consisting of G61E, E631K, G446E, P630L, E628K, M836I, D837N, K876X, G1003R, Vl 1541, G1296R, P1279S, P1279L, P1279F, H1477Y, I1115T, R427Stop, P459L, G1272V, G61E, E365K, G446E,

E1042K, V1126M, D1248N, W1444Stop, V1565I, P1690F, and R1798Stop and combinations thereof. Optionally, the substitution is I1115T. Optionally, the substitution is G1272V. Optionally, the substitution is R427Stop. Optionally, the substitution is P459L.

Optionally, the determining step includes detecting one or more mutations in a nucleic acid encoding PTPRD. Optionally, the mutation is a substitution selected from the group consisting of G182A, G1891A, G1337A, C1889T, G1882A, G2507A,

G2510A, A2626T, G3007A, G3460A, G3886A, G3835T, C3836T, C4429T, T3486C, C1421T, C1518T, G3957T, C1421T, C1518T, G3957T, G324A, G1235A, G1479A, G3266A, G3518A, G3884A, G4474A, G4475A, G4835A, C5210T, C5211T, C5533T, and C5534T and combinations thereof. Optionally, the substitution is T3486C. Optionally, the substitution is C 1421 T. Optionally, the substitution is G3957T.

Optionally, the substitution is T3486C and C1518T.

Also provided are methods of determining whether a subject has or is at risk for developing a melanoma, glioblastoma, Ewing's sarcoma, or primitive neuroectodermal tumor. The method comprises obtaining a biological sample from the subject and determining in the sample whether the nucleic acid encoding PTPRD comprises one or more mutations, wherein the mutation is a substitution selected from the group consisting of G182A, G1891A, G1337A, C1889T, G1882A, G2507A, G2510A, A2626T, G3007A, G3460A, G3886A, G3835T, C3836T, C4429T, T3486C, C1421T, C1518T, G3957T, C1421T, C1518T, G3957T, G324A, G1235A, G1479A, G3266A, G3518A, G3884A, G4474A, G4475A, G4835A, C5210T, C5211T, C5533T, and C5534T. Optionally, the the subject has or is at risk for developing melanoma and the substitution is selected from the group consisting of G182A, G1891A, G1337A, C1889T, G1882A, G2507A, G2510A, A2626T, G3007A, G3460A, G3886A, G3835T, C3836T, C4429T, T3486C, G324A, G1235A, G1479A, G3266A, G3518A, G3884A, G4474A, G4475A, G4835A, C5210T, C5211T, C5533T, and

C5534T. Optionally the substitution is T3486C.

Described herein is an inherited mutation in the PTPRD gene that has been identified in association with cancer or risk for developing cancer. Specifically, as described in the examples below, an inherited mutation in PTPRD was identified at nucleotide 3486 of SEQ ID NO: 1 in a glioblastoma sample. For example, the mutation is a thymine to cytosine base substitution. The thymine to cytosine base substitution alters residue 1115 of SEQ ID NO:2, resulting in an isoleucine to threonine amino acid substitution.

Further provided is a method of determining whether a subject has or is at risk for developing cancer. The method comprises obtaining a biological sample from the subject, and determining in the sample whether PTPRD comprises an inherited mutation. An inherited mutation indicates the subject has or is at risk for developing cancer. Optionally, the inherited mutation of PTPRD comprises a substitution or deletion at nucleotide position 3486 of SEQ ID NO:1, including, for example, the substitution T3486C. Optionally, the substitution in the PTPRD nucleic acid sequence results in an amino acid substitution at residue 1115 of SEQ ID NO:2. Optionally, the amino acid substitution is Il 115T. Optionally, the method includes determining in the sample whether PTPRD comprises an amino acid substitution at residue 1115 of SEQ ID NO:2. A substitution indicates the subject has or is at risk of developing cancer. Optionally, the amino acid substitution is I1115T. The inherited mutation of PTPRD can, for example, result in decreased expression or decreased activity of PTPRD as compared to a control. As used herein, the term decreased expression or decreased activity is compared to a control. A decrease or lower level in expression or activity of PTPRD as compared to a control means that the level of expression or activity of PTPRD is at least 1.5 times lower in the biological sample from a subject being tested than in a control sample. As used throughout, lower or decrease as compared to a control refer to decreases below a control. As used herein, control refers to a reference standard from, for example, an untreated or normal (wild-type) sample or subject.

The term inherited mutation, as used herein, includes a mutation that is transmitted through the germ line from parental DNA to progeny DNA. As used herein, the term inherited mutation, also refers to a mutation that is present in the germline of a subject, i.e., in the sperm or eggs of a subject, but may or may not be present in the subject's parental DNA. The term inherited mutation also includes a mutation that is present in non-neoplastic cells of a subject. As used throughout, the term mutation includes one or more deletions, insertions, or substitutions of one or more amino acids or nucleotides. Thus, in the provided methods, the mutation can be a deletion, insertion, or substitution. Optionally, the mutation is a deletion or substitution. Optionally, the mutation is a substitution. By way of example, an insertion or deletion can result in an alteration of the reading frame of the gene, which alters the function of the gene. A point mutation or substitution can, for example, result in a mutation, e.g., a missense mutation, or a nonsense mutation, that alters the function of a gene. For example, the function of a gene can be altered in that the gene is no longer transcribed at wild-type levels. Alternatively, the amino acid sequence encoded by the gene no longer functions at control levels.

The sample can be any biological sample and can comprise a non-neoplastic sample or a neoplastic sample. As used herein a biological sample is a sample derived from a subject and includes, but is not limited to, any cell, tissue or biological fluid. For example, the sample can be a tissue biopsy, blood or components thereof, bone marrow, urine, saliva, tissue infiltrate and the like. The biological fluid may be a cell culture medium or supernatant of cultured cells from a subject Optionally, the biological sample contains cerebral spinal fluid. Optionally, the biological sample contains non-neoplastic cells.

Optionally, a genetic sample is obtained. The genetic sample comprises a nucleic acid, preferably RNA and/or DNA. A genetic sample may be obtained using any known technique including those described in Ausubel et al., Current Protocols in Molecular Biology (John Wiley & Sons, Inc., New York, 1999); Molecular Cloning: A

Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (Cold Spring Harbor Laboratory Press: 1989); and Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984). The nucleic acid may be purified from whole cells using DNA or RNA purification techniques. The genetic sample may also be amplified using PCR or in vivo techniques requiring subcloning. The genetic sample can be obtained by isolating mRNA from the cells of the biological sample and reverse transcribing the RNA into DNA in order to create cDNA (Khan et al. Biochem. Biophys. Acta 1423:17 28, 1999).

The genetic sample can be analyzed for the presence or absence of a particular mutation. Thus, determining whether the PTPRD nucleic acid comprises an inherited mutation can, for example, be carried out by a method selected from the list consisting of sequencing, PCR, RT-PCR, quantitative PCR, one step PCR, restriction fragment length polymorphism, hybridization techniques, Northern blot, microarray technology, gene chip, in situ hybridization, DNA microarray technology, and the like. Alternatively, determining whether the PTPRD amino acid sequence comprises an inherited mutation can, for example, be carried out by Western Blot or protein sequencing. The analytical techniques to determine whether the PTPRD nucleic acid or amino acid sequences comprise an inherited mutation are known. See., e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Press, Cold Spring Harbor, NY (2001).

Also provided herein is a method of treating or preventing cancer, for example, melanoma, Ewing's sarcoma, primitive neuroectodermal tumor or glioblastoma in a subject. Optionally, the cancer is brain cancer, lung cancer, breast cancer, prostate cancer, colon cancer, stomach cancer, pancreatic cancer, bone cancer, soft tissue cancer, and skin cancer. Optionally, the skin cancer is melanoma. Optionally, the type of cancer can be brain cancer, e.g., a glioblastoma. Optionally, the type of cancer can be bone cancer (e.g., Ewing's sarcoma) or soft tissue cancer.

Optionally, the cancer is a primitive neuroectodermal tumor.

The method comprises administering a composition comprising a pharmaceutically acceptable excipient and PTPRD or a nucleic acid sequence encoding PTPRD to the subject. PTPRD is used by way of example herein, but homo logs, variants and fragments could be used in the methods of treating or preventing cancer and compositions used the methods, as long as the homologs, variants and fragments are functionally equivalent to PTPRD. Thus, the PTPRD used in the compositions and methods of treatment includes homologs, variants and fragments that do not alter the nature or function of PTPRD. Provided herein are compositions containing the provided polypeptides and nucleic acid molecules and a pharmaceutically acceptable carrier described herein. Pharmaceutical compositions comprising one or more of the provided molecules (i.e., polypeptides and nucleic acids) herein may include pharmaceutical carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agent, and the like. The compositions of the present application can be administered in vivo in a pharmaceutically acceptable carrier. By pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable. Thus, the material may be administered to a subject, without causing undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.

The disclosed compositions can be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Thus, the disclosed compositions can be administered, for example, orally, parenterally (e.g., intravenously), intraventricularly, intramuscularly, intraperitoneally, transdermally, extracorporeally, or topically. The compositions can be administered locally (e.g., into a tumor).

The polypeptides or nucleic acids may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (21th ed.) ed. David B. Troy, Lippincott Williams & Wilkins, 2005. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8.5, and more preferably from about 7.8 to about 8.2. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of composition being administered.

The compositions can be formulated to ensure that they cross the blood brain barrier (BBB), as necessary. They can be formulated, for example, in liposomes. The liposomes may comprise one or more moieties which are selectively transported into specific cells or organs (targeting moieties), thus providing targeted drug delivery. Exemplary targeting moieties include folate, biotin, mannosides, antibodies, surfactant protein A receptor and gpl20. To ensure that agents of the invention cross the BBB, they may also be coupled to a BBB transport vector (see Bickel, et al., Adv. Drug Delivery Reviews, vol. 46, pp. 247-279, 2001). Exemplary transport vectors include cationized albumin or the 0X26 monoclonal antibody to the transferrin receptor; these proteins undergo absorptive -mediated and receptor-mediated transcytosis through the BBB, respectively.

Examples of other BBB transport vectors that target receptor-mediated transport systems into the brain include factors such as insulin, insulin-like growth factors (IGF-I, IGF-II), angiotensin II, atrial and brain natriuretic peptide (ANP,

BNP), interleukin I (IL-I) and transferrin. Monoclonal antibodies to the receptors which bind these factors may also be used as BBB transport vectors. BBB transport vectors targeting mechanisms for absorptive-mediated transcytosis include cationic moieties such as cationized LDL, albumin or horseradish peroxidase coupled with polylysine, cationized albumin or cationized immunoglobulins. Small basic oligopeptides such as the dynorphin analogue E-2078 and the ACTH analogue ebiratide can also cross the brain via absorptive -mediated transcytosis and are potential transport vectors.

Other BBB transport vectors target systems for transporting nutrients into the brain. Examples of such BBB transport vectors include hexose moieties such as, for example, glucose; monocarboxylic acids such as, for example, lactic acid; neutral amino acids such as, for example, phenylalanine; amines such as, for example, choline; basic amino acids such as, for example, arginine; nucleosides such as, for example, adenosine; purine bases such as, for example, adenine, and thyroid hormones such as, for example, triiodothyridine. Antibodies to the extracellular domain of nutrient transporters can also be used as transport vectors. In some cases, the bond linking the agent to the transport vector may be cleaved following transport into the brain in order to liberate the biologically active compound. Exemplary linkers include disulfide bonds, ester-based linkages, thioether linkages, amide bonds, acid- labile linkages, and Schiff base linkages. Avidin/biotin linkers, in which avidin is covalently coupled to the BBB drug transport vector, may also be used. Avidin itself may be a drug transport vector. Optionally, the nucleic acid molecule or polypeptide is administered by a vector comprising the nucleic acid molecule or a nucleic acid sequence encoding the polypeptide. There are a number of compositions and methods which can be used to deliver the nucleic acid molecules and/or polypeptides to cells, either in vitro or in vzVo via, for example, expression vectors. These methods and compositions can largely be broken down into two classes: viral based delivery systems and non- viral based deliver systems. Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein.

As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids into the cell without degradation and include a promoter yielding expression of the nucleic acid molecule and/or polypeptide in the cells into which it is delivered. Viral vectors are, for example, Adenovirus, Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus, Sindbis, and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors, in general are described by Coffin et al, Retorviruses, Cold Spring Harbor Laboratory Press (1997), which is incorporated by reference herein for the vectors and methods of making them. The construction of replication-defective adenoviruses has been described (Berkner et al., J. virology 61 :1213-20 (1987); Massie et al., MoI. Cell. Biol. 6:2872-83 (1986); Haj-Ahmad et al., J. virology 57:267-74 (1986);

Davidson et al., J. Virology 61 :1226-39 (1987); Zhang et al., BioTechniques 15:868- 72 (1993)). The benefit and the use of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types, since they can replicate within an initial infected cell, but are unable to form new infections viral particles. Recombinant adenoviruses have been shown to achieve high efficiency after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and a number of other tissue sites. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.

The provided polypeptides and/or nucleic acid molecules can be delivered via virus like particles. Virus like particles (VLPs) consist of viral protein(s) derived from the structural proteins of a virus. Methods for making and using virus like particles are described in, for example, Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

The provided polypeptides can be delivered by subviral dense bodies (DBs). DBs transport proteins into target cells by membrane fusion. Methods for making and using DBs are described in, for example, Pepperl-Klindworth et al., Gene Therapy

10:278-84 (2003).

The provided polypeptides can be delivered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO 2006/110728. Non- viral based delivery methods can include expression vectors comprising nucleic acid molecules and nucleic acid sequences encoding polypeptides, wherein the nucleic acids are operably linked to an expression control sequence. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from such corporations as Novagen

(Madison, WI), Clonetech (Palo Alto, CA), Stratagene (La Jolla, CA), and Invitrogen/Life Technologies (Carlsbad, CA). Vectors typically contain one or more regulatory regions. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3 ' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, and introns.

Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis B virus, cytomegalovirus (CMV), from heterologous mammalian promoters (e.g. β-actin promoter or EFl α promoter) or from hybrid or chimeric promoters (e.g., CMV promoter fused to the β-actin promoter). Promoters from the host cell or related species are also useful herein. Enhancer generally refers to a sequence of DN A that functions at no fixed distance from the transcription start site and can be either 5 ' or 3 ' to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. They are usually between 10 and 300bp in length, and they function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes

(globin, elastase, albumin, fetoprotein, and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression. Preferred examples are the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

The promoter and/or the enhancer can be inducible (e.g. chemically or physically regulated). A chemically regulated promoter and/or enhancer can, for example, be regulated by the presence of alcohol, tetracycline, a steroid, or a metal. A physically regulated promoter and/or enhancer can, for example, be regulated by environmental factors, such as temperature and light. Optionally, the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize the expression of the region of the transcription unit to be transcribed. In certain vectors, the promoter and/or enhancer region can be active in a cell type specific manner. Optionally, in certain vectors, the promoter and/or enhancer region can be active in all eukaryotic cells, independent of cell type. Preferred promoters of this type are the

CMV promoter, the SV40 promoter, the β-actin promoter, the EF lα promoter, and the retroviral long terminal repeat (LTR).

The vectors also can include, for example, origins of replication and/or markers. A marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell. The marker product is used to determine if the vector has been delivered to the cell and once delivered is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure.

Examples of other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag™ tag (Kodak; New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus.

The method of treating or preventing cancer, for example, glioblastoma, Ewing's sarcoma, primitive neuroectodermal tumor or melanoma, in a subject can further comprise administering to the subject a therapeutic agent or radiation therapy or a combination thereof.

The therapeutic agent can, for example, be a chemotherapeutic agent. A chemotherapeutic agent is a compound or composition effective in inhibiting or arresting the growth of an abnormally growing cell. Thus, such an agent may be used therapeutically to treat cancer as well as other diseases marked by abnormal cell growth. Illustrative examples of anti-cancer compounds include, but are not limited to, bexarotene, gefϊtinib, erlotinib, gemcitabine, paclitaxel, docetaxel, topotecan, irinotecan, vinorelbine, capecitabine, leucovorin, oxaliplatin, bevacizumab, cetuximab, panitumumab, bortezomib, oblimersen, hexamethylmelamine, ifosfamide, CPT-Il, deflunomide, cycloheximide, dicarbazine, asparaginase, mitotant, vinblastine sulfate, carboplatin, colchicine, etoposide, melphalan, 6-mercaptopurine, teniposide, vinblastine, antibiotic derivatives (e.g. anthracyclines such as doxorubicin, liposomal doxorubicin, and diethylstilbestrol doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil (FU), 5 -FU, methotrexate, floxuridine, interferon alpha-2B, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,

BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, vincristine and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., mephalen, chlorambucil, mechlorethamine (nitrogen mustard) and thiotepa); and steroids (e.g., bethamethasone sodium phosphate). Optionally, the chemotherapeutic agent is temozolomide or carmustine.

Any of the aforementioned therapeutic agents can be used in any combination with the compositions described herein. Combinations are administered either concomitantly (e.g., as an admixture), separately but simultaneously (e.g., via separate intravenous lines into the same subject), or sequentially (e.g., one of the compounds or agents is given first followed by the second). Thus, the term combination is used to refer to either concomitant, simultaneous, or sequential administration of two or more agents. According to the methods taught herein, the subject is administered an effective amount of the composition and/or agent. The terms effective amount and effective dosage are used interchangeably. The term effective amount is defined as any amount necessary to produce a desired physiologic response. Effective amounts and schedules for administering the agent may be determined empirically, and making such determinations is within the skill in the art. The dosage ranges for administration are those large enough to produce the desired effect in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex, type of disease, the extent of the disease or disorder, route of administration, or whether other drugs are included in the regimen, and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosages can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products.

As used throughout, the terms peptide, polypeptide, or protein are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used herein interchangeably to refer to amino acid sequences. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids comprising the molecule and that a peptide of the invention can contain up to several amino acid residues or more. As used throughout, subject can be a vertebrate, more specifically a mammal (e.g. a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and any other animal. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. As used herein, patient or subject may be used interchangeably and can refer to a subject with a disease or disorder (e.g. cancer). The term patient or subject includes human and veterinary subjects.

As used herein the terms treatment, treat, or treating refers to a method of reducing the effects of a disease or condition or symptom of the disease or condition. Thus in the disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%,

60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method for treating a disease is considered to be a treatment if there is a 10% reduction in one or more symptoms or signs (e.g., size of the tumor or rate of tumor growth) of the disease in a subject as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%,

50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition. As used herein, the terms prevent, preventing, and prevention of a disease or disorder refers to an action, for example, administration of a composition or therapeutic agent, that occurs before or at about the same time a subject begins to show one or more symptoms of the disease or disorder, which inhibits or delays onset or severity of one or more symptoms of the disease or disorder. As used herein, references to decreasing, reducing, or inhibiting include a change of 10%, 20%, 30%,

40%, 50%, 60%, 70%, 80%, 90% or greater as compared to a control level. Such terms can include but do not necessarily include complete elimination.

Disclosed are materials, compositions, and components that can be used for, can be used in conjunction with, can be used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutations of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a method is disclosed and discussed and a number of modifications that can be made to a number of molecules including the method are discussed, each and every combination and permutation of the method, and the modifications that are possible are specifically contemplated unless specifically indicated to the contrary. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed, it is understood that each of these additional steps can be performed with any specific method steps or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is specifically contemplated and should be considered disclosed. Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application.

A number of aspects have been described. Nevertheless, it will be understood that various modifications may be made. Furthermore, when one characteristic or step is described it can be combined with any other characteristic or step herein even if the combination is not explicitly stated. Accordingly, other aspects are within the scope of the claims.

EXAMPLES

Example 1. Mutational Inactivation of PTPRD in Glioblastoma Multiforme and Malignant Melanoma Materials and Methods

Tumor tissues. A panel of 21 glioblastoma multiforme (GBM) cell lines were obtained from the American Type Culture Collection (U87MG, U138MG, M059J, Hs683, H4, A172, LN18, LN229, CCF-STTGl, T98G, DBTRG-05MG), DSMZ (8MGBA, 42MGBA, DKMG, GAMG, GMSlO, LN405, SNB19), and the Japan Health Sciences Foundation Health Science Research Resources Bank (AM38, NMC-

Gl, KG-I-C). Normal human astrocytes (NHAs) were obtained from Clonetics and Allcells (Emeryville, CA). All cell lines were growth in DMEM + 10% FBS at 37° in 5% CO2.

Subcutaneous xenografts in immunodeficient mice were obtained from the Duke University Brain Tumor Center or created in the Lombardi Comprehensive Cancer Center Animal Shared Resource from tissue taken from patients undergoing craniotomy at Georgetown University Hospital (IRB #2006-344).

Snap frozen primary GBM tumors and paired blood samples were obtained from the Brain Tumour Tissue Bank (London Health Sciences Centre; Ontario, Canada) funded by the Brain Tumour Foundation of Canada. All tumors were graded by a neuropathologist as good or moderate on a scale of good to poor depending on the amount of tumor cells present (as opposed to hemorrhagic, necrotic, or fibrous tissue). All tumor samples were further categorized as "tumor center."

A panel of 10 primary GBM cell cultures were derived from primary tumor samples at time of surgical resection at the University of Iowa Medical Center by dissociation with collagenase and then cultured in DMEM/F12 containing 15% FBS,

10 μg/mL insulin, and 5 ng/niL bFGF at 37° in 5% CO2.

A panel of 47 malignant melanoma tumor and paired blood samples were collected during surgical resection at the National Cancer Institute. The primary cell cultures 16T and 86T used for functional analysis were derived from melanoma tumor samples by dissociation with collagenase and then cultured in RPMI + 10% FBS at

37° in 5% CO2.

Microarrays and bioinformatics. Genomic DNA derived from GBM tumor samples was interrogated with Affymetrix 250KNsp I Human Gene Chip Arrays using protocols described by the manufacturer. Data processing was performed using dCHIP (Li et al, Proc. Natl. Acad. Sci. USA 98:31-6 (2001)). The scanned array images and processed data sets have been deposited in the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo).

Western blot. Primary antibodies used were PTPRD clone C-18 (Santa Cruz Biotechnology; Santa Cruz, CA) and α-tubulin Ab-2 clone DMlA (Neomarkers). DNA sequencing. Individual exons of PTPRD were PCR amplified from genomic DNA using conditions and primer pairs described by Sjoblom et al. (Sjoblom et al., Science 314:268-74 (2006)). PCR products were purified using the Exo/SAP method followed by a Sephadex spin column. Sequencing reactions were performed using Big Dye v3.1 (Applied Biosystems; Foster City, CA) using an M13F primer, and analyzed on an Applied Biosystems 3730XL capillary sequencer. Sequences were analyzed using Mutation Surveyor (Softgenetics; State College, PA). Traces with putative mutations were re-amplified and sequenced from both tumor and matched normal DNA.

PTPRD Lentivirus. A wild-type PTPRD cDNA (MGC 119751) was obtained from Open Biosystems (Huntsville, AL) and cloned into the pCDFl-MCS2-EFl-Puro lentiviral expression vector backbone (System Biosciences; Mountain View, CA). To make virus, this construct was co-transfected into 293T cells with pVSV-G (Addgene;

Cambridge, MA) and pFIV-34N (System Biosciences; Mountain View, CA) helper plasmids using Fugene 6 (Roche; Indianapolis, IN) as described by the manufacturer. Virus-containing conditioned media was harvested 48 hours after transfection, filtered, and used to infect recipient cells in the presence of 8 ug/mL polybrene. Site-directed mutagenesis. Mutations identified in GBM and melanoma tumors were engineered into the pCDFl -PTPRD construct by site-directed mutagenesis using the QuikChange II XL kit (Stratagene; La Jolla, CA) as directed by the manufacturer. The coding sequence of all expression vectors was verified by DNA sequencing. Flow cytometry. Cells were pulsed with 10 μmol/L BrdU for 1 hour, trypsinized, and centrifuged. Cells were fixed and stained using the BrdU Flow Kit (PharMingen) and analyzed by flow cytometry in a BD FAC Sort instrument using FCS Express v.3 software (DeNovo Software; Los Angeles, CA).

Apoptosis quantification assay. Cells were collected by trypsinization, centrifuged, and simultaneously fixed and stained in a solution containing 3.7% formaldehyde, 0.5% Igepal, and 10 μg/mL Hoechst 33258 in PBS. Fluorescence microscopy was used to visualize and score apoptotic nuclei. At least 200 cells were counted for each determination in triplicate.

Microscopy. All imaging was performed on an Olympus BX61 light microscope (Olympus; Center Valley, PA) with a 4OX Plan-Apochromat objective. Statistical Analysis. Two-tailed unpaired t-test analysis of BrdU incorporation data was performed using GraphPad Prism software (GraphPad Software; La Jolla, CA).

Identification of mutations in Protein Tyrosine Phosphatase Receptor D (PTPRD) in globlastoma multiforme (GBM). In an effort to discover genes that contribute to the pathogenesis of GBM, Affymetrix 250K Gene Chip Arrays were used to identify recurrent copy number alterations in a panel of 58 GBM tumor samples (Fig. IA). Focal deletions of the PTPRD gene on chromosome 9p23-24.1 were among the most prevalent deletions detected, present in 14% of the GBM samples studied (Fig. IB and Table 1). This frequency of focal deletion is higher than that of PTEN (9%) and similar to that of CDKN2C (also named pi 8INK4c, 16%), a recently identified GBM tumor suppressor gene (Solomon et al., Cancer Research 68:2564-9 (2008); Wiedermeyer et al., Cancer Cell 13:355-64 (2008)). Larger scale loss of the PTPRD gene was present in an additional 33% of the samples (Fig. 1C and Table 2). Intriguingly, several studies have suggested the presence of another important tumor suppressor gene on chromosome 9p telomeric to the CDKN2A/B locus in tumor types including astrocytoma, melanoma, and lung adenocarcinoma (Ueki et al., Hum. MoI. Genet. 3:1841-5 (1994); Puig et al., Am. J. Hum. Genet. 57:395-402 (1995); Kim et al., Cancer Research 57:400-3 (1997); Pollock et al., Cancer Research 61 :1154-61 (2001); Cook et al., Int. J. Cancer 93:361-7 (2001)).

Therefore, PTPRD was considered to be an attractive candidate as a GBM tumor suppressor gene, and possibly relevant to a range of other tumor types as well.

Table 1 : Focal deletions (<10 Megabases (Mb)) of the PTPRD gene identified in 8 out of 58 glioblastoma multiforme (GBM) tumor samples by Affymetrix 250K SNP array (Affymetrix; Santa Clara, CA).

Figure imgf000023_0001

Based on hgl8 genome assembly.

Table 2: Large-scale chromosomal loss (>10Mb) encompassing the PTPRD gene identified in 21 out of 58 GBM tumor samples by Affymetrix 250K SNP array (Affymetrix; Santa Clara, CA).

Figure imgf000024_0001

Based on hgl8 genome assembly.

Identification of inherited mutation in PTPRD in patient with GBM history. To determine if PTPRD is genetically altered by mutation during GBM tumorigenesis, the 35 coding exons of the PTPRD gene were sequenced in tumor samples lacking focal deletions of PTPRD and in corresponding normal tissue. This sequence analysis identified somatic mutations of the PTPRD gene in three samples, including two missense mutations and one nonsense mutation (Fig. 2A and 2C). Additionally, a heterozygous germline mutation was identified that was accompanied by somatic loss of the wild-type allele in the tumor of a GBM patient with a history of multiple primary malignancies (Fig. 2A and 2B). This mutation is not a reported sequence nucleotide polymorphism (SNP), and was not present in any of > 100 alleles of

PTPRD sequenced during the course of this study. Together, these data demonstrate that PTPRD is altered by somatic mutation during GBM pathogenesis.

Identification of mutations in PTPRD in melanoma tumor samples. To determine if mutations of PTPRD were present in a second tumor type proposed to harbor an additional 9p tumor suppressor gene, PTPRD was sequenced in 47 melanoma tumor samples. Two somatic nonsense mutations and eight somatic missense mutations were identified (Table 3, Fig. 3, and Fig. 4) in a total of seven samples. All of these mutations were C/G>T/A trans versions, consistent with UV-induced DNA damage. Additionally, three of the mutations were dinucleotide CC>TT mutations caused by the formation of UV-induced cyclobutane pyrimidine dimers (Fig. 4). Three of the seven samples harboring somatic mutations of PTPRD displayed loss of heterozygosity (LOH) of the wild-type allele. Furthermore, tumor 76T harbored four independent mutations of the gene, increasing the likelihood that both alleles of the gene had been targeted by mutation in this sample. Five (5) of these 7 samples with PTPRD mutation also harbor activating mutations of either B-Raf or N-Ras. This

12% mutation frequency makes PTPRD among the most commonly mutated genes in sporadic melanoma reported to date, which include B-Raf (-60%), p53 (0-25%), N- Ras (10-15%), PTEN (-10%), pl6INK4a (0-5%), and PIK3CA (<1%) (Fecher et al, J. Clin. Oncol. 25:1606-20 (2007); Curtin et al., N. Engl. J. Med. 355:2135-47 (2005)). The 14 mutations of PTPRD reported here are distributed roughly evenly throughout the various extracellular and intracellular domains of the encoded PTPRD protein (Fig. 5), though there appears to be a mini-hotspot in the first and second fibronectin type-III repeat. Table 3: Identification of somatic mutations of PTPRD in malignant melanoma. Ten somatic mutations of PTPRD were identified in 7 out of 47 malignant melanoma tumor samples.

Figure imgf000026_0001

ΛGenomic position is based on the hgl8 genome assembly. #Transcript

ENST00000381196 was used for annotation of the nucleotide and amino acid changes. * Assignment of functional domains was based on UniProtKB/Swiss-Prot P23468-1. Ig_C2, immunoglobulin- like C2-type domain; FN III, fibronectin type-Ill domain; PTPc, protein tyrosine phosphatase catalytic domain.

Expression of PTPRD results in growth suppression and increased apoptosis.

Despite its potential importance, functional data implicating PTPRD deletion or mutation in tumorigenesis are lacking. To determine if PTPRD has the growth suppressing properties expected of a broad spectrum tumor suppressor gene, the functional consequences of reconstituting PTPRD expression in GBM and melanoma cells were examined. A 5.1 kilobase human PTPRD cDNA was obtained, cloned into a lentiviral expression vector, and packaged into an infectious lentivirus. Infection of H4 cells that harbor biallelic deletion of PTPRD (Fig. IB and Table 1) with lenti- PTPRD led to expression of both the PTPRD proprotein and its mature cleavage products (which then reassemble at the cell membrane to form a heterodimer) (Pulido et al, J. Biol. Chem. 270:6722-8 (1995)) (Fig. 6A). Infection of H4 cells with lenti- PTPRD but not vector alone led to a transient growth arrest evidenced by a reduction in BrdU incorporation and an increase in both Gl and sub-Gl cell populations (Fig. 6B). Infection with lenti-PTPRD had a similar effect on 8MGB A cells, which also harbor a focal deletion of PTPRD (Fig. IB).

Two primary melanoma cell cultures harboring homozygous missense mutations of PTPRD (16T and 86T with G446E in the second FN III domain and V1565I in the first PTPc domain, respectively) were also infected with lenti-PTPRD. Infection of both primary cell cultures with wild-type PTPRD but not vector alone led to significant growth inhibition and decrease in cell viability (Fig. 6C), as well as a substantial, time-dependent increase in apoptotic cells (Fig. 6D). These are the first reported data indicating that PTPRD has growth suppressive properties when expressed in human cancer cells, supporting the hypothesis that PTPRD is a human tumor suppressor gene.

The consequences of tumor-derived mutations on PTPRD function in these assays were additionally examined. To do this, five tumor-derived mutations were introduced into lenti-PTPRD, including two mutations in the second FN-III domain mini-hotspot (one each from GBM (P459L) and melanoma(G446E)), one mutation in the first PTPc domain (melanoma (V1565I)), and two mutations flanking the proprotein cleavage site (one each from GBM (Il 115T) and melanoma(D1248N)). Initially, H4 cells were infected with wild-type and mutant lenti-PTPRD, protein lysates prepared, and PTPRD expression examined by Western blot. As shown in Fig. 7A, infection of H4 cells with lentivirus expressing either wild-type or mutant PTPRD resulted in similar levels of protein expression. However, there was a marked decrease in growth inhibition as measured by BrdU incorporation, indicating that each of the five tumor-derived mutants alleviated the growth suppression activity of PTPRD, albeit to differing extents (Fig. 7B). Next, 16T melanoma cells were similarly infected with wild-type and mutant PTPRD lentiviruses. As depicted in Fig. 7C, wild-type PTPRD led to apoptosis of approximately 75% of the cells at 10 days post-infection, whereas the mutant PTPRD lentiviruses led to a substantially reduced fraction of cells that had undergone programmed cell death. When taken together, these experiments demonstrate that tumor-derived mutations of PTPRD attenuate its function, confirming that the mutations of PTPRD are likely to be pathogenic. Example 2. Identification of Additional Mutations in PTPRD in Malignant Melanoma

Seventy-nine (79) melanoma cancer samples were analyzed as described for Example 1 and the results are shown below in Table 4. Eighteen mutations were identified affecting 20.3% of the tumors analyzed. The nucleotide and amino acid positions are relative to the PTPRD sequence of Accession No. NM 002839.2 in the GenBank Accession Database.

Table 4. Mutations identified in PTPRD

Figure imgf000029_0001

Nucleotide and amino acid change resulting from mutation. When multiple mutations in the same gene in a tumor were observed, the mutations are separated by a comma. "X" refers to stop codon. "LOH" refers to cases wherein the wild-type allele was lost and only the mutant allele remained. "Splice site" refers to a case wherein the alteration affected 15 bases spanning the exon.

2 Abbreviations for the functional domains: FN3: Fibronectin Type III; Ig-like C2-type 1 : immunoglobulin-like domain containing two cysteine residues; PTPc: protein tyrosine phosphatase domain of PTPs containing single catalytic domains; TM: transmembrane domain.

3Mutations previously observed in NRAS, BRAF. "None" refers no mutation observed.

Claims

WHAT IS CLAIMED IS:
1. A method of determining whether a subject has or is at risk for developing cancer, comprising: a. obtaining a biological sample from the subject; and b. determining in the sample whether protein tyrosine phosphatase receptor type D (PTPRD) comprises one or more mutations, wherein one or more mutations in PTPRD indicates the subject has or is at risk for developing a cancer selected from the group consisting of glioblastoma, melanoma, primitive neuroectodermal cancer, bone cancer, and soft tissue sarcoma.
2. The method of claim 1, wherein the sample is a neoplastic sample.
3. The method of claim 1, wherein the sample is a non-neoplastic sample.
4. The method of claim 1, wherein the mutation is a substitution or a deletion.
5. The method of claim 1, wherein the determining step includes detecting one or more mutations in a nucleic acid encoding PTPRD.
6. The method of claim 5, wherein the mutation is a substitution selected from the group consisting of G182A, G1891A, G1337A, C1889T, G1882A, G2507A, G2510A, A2626T, G3007A, G3460A, G3886A, G3835T, C3836T, C4429T, T3486C, C1421T, C1518T, G3957T, C1421T, C1518T, G3957T, G324A, G1235A, G1479A, G3266A, G3518A, G3884A, G4474A, G4475A, G4835A, C5210T, C5211T, C5533T, C5534T, and combinations thereof.
7. The method of claim 6, wherein the substitution is T3486C.
8. The method of claim 6, wherein the substitution is C1421T, C1518T, or G3957T.
9. The method of claim 6, wherein the substitution is C1421T, C1518T, G3957T, G324A, G1235A, G1479A, G3266A, G3518A, G3884A, G4474A, G4475A, G4835A, C5210T, C521 IT, C5533T, or C5534T.
10. The method of claim 1, wherein the determining step includes detecting one or more mutations in the amino acid sequence of PTPRD.
11. The method of claim 10, wherein the mutation is a substitution selected from the group consisting of G61E, E631K, G446E, P630L, E628K, M836I, D837N, K876X, G1003R, Vl 1541, G1296R, P1279S, P1279L, P1279F, H1477Y, Il 115T, R427Stop, P459L, G1272V, G61E, E365K, G446E, E1042K, V1126M, D1248N, W1444Stop, V1565I, P1690F, R1798Stop, and combinations thereof.
12. The method of claim 11, wherein the substitution is Il 115T.
13. The method of claim 11, wherein the substitution is R427Stop, P459L, or
G 1272V.
14. The method of claim 11, wherein the substitution is G61E, E365K, G446E, E1042K, V1126M, D1248N, W1444Stop, V1565I, P1690F, or R1798Stop.
15. The method of any one of claims 1-14, wherein the sample is a tissue biopsy.
16. The method of any one of claims 1-14, wherein the sample comprises cerebral spinal fluid.
17. A method of treating or preventing cancer in a subject comprising administering a composition comprising a pharmaceutically acceptable excipient and PTPRD or a nucleic acid sequence encoding PTPRD to the subject, wherein the cancer is selected from the group consisting of glioblastoma, melanoma, primitive neuroectodermal cancer, bone cancer, and soft tissue sarcoma.
18. The method of claim 17, wherein the composition comprises a nucleic acid sequence encoding PTPRD.
19. The method of claim 18, wherein a vector comprises the nucleic acid sequence encoding PTPRD.
20. The method of claim 19, wherein the vector is a viral vector.
21. The method of any one of claims 17-20, further comprising administering to the subject a therapeutic agent or radiation therapy or a combination thereof.
22. The method of claim 21, wherein the therapeutic agent is a chemotherapeutic agent.
23. A method of determining whether a subject has or is at risk for developing cancer, comprising determining whether the subject has an inherited mutation in PTPRD, wherein the inherited mutation indicates the subject has or is at risk for developing cancer.
24. The method of claim 23, wherein an inherited mutation of PTPRD results in decreased expression or activity of PTPRD.
25. The method of claim 23, wherein the inherited mutation of PTPRD comprises a substitution or deletion at nucleotide position 3486 of SEQ ID NO: 1.
26. The method of claim 25, wherein the substitution is T3486C.
27. The method of claim 25, wherein the substitution in the PTPRD nucleic acid sequence results in an amino acid substitution or deletion of residue 1115 of SEQ ID NO:2.
28. The method of claim 27, wherein the amino acid substitution is Il 115T.
29. The method of claim 23, wherein the determining step is carried out by a method selected from the list consisting of PCR, RT-PCR, and DNA sequencing.
30. A method of determining whether a subject has or is at risk for developing cancer, comprising: a. obtaining a sample from the subject; and b. determining in the sample whether PTPRD comprises a mutation, wherein the mutation is an amino acid substitution or deletion of residue 1115 of SEQ ID NO:2 or wherein the mutation is a substitution or deletion at nucleotide position 3486 of SEQ ID NO:1, wherein a mutation indicates the subject has or is at risk for developing cancer.
31. The method of claim 30, wherein the mutation is a substitution and the substitution is T3486C.
32. The method of claim 30, wherein the mutation is an amino acid substitution and the amino acid substitution is Il 115T.
33. The method of claim 30, wherein the determining step is carried out by Western blot.
34. The method of any one of claims 23-33, wherein the type of cancer is selected from a list consisting of brain cancer, lung cancer, breast cancer, prostate cancer, colon cancer, stomach cancer, pancreatic cancer, soft tissue cancer, primitive neuroectodermal cancer, bone cancer, and skin cancer.
35. The method of claim 34, wherein the brain cancer is glioblastoma.
36. The method of any one of claims 23-33, wherein the biological sample is a tissue biopsy.
37. The method of any one of claims 23-33, wherein the biological sample comprises cerebral spinal fluid.
38. The method of any one of the claims 23-33, wherein the biological sample is a non-neoplastic sample.
PCT/US2009/067647 2008-12-12 2009-12-11 Methods of diagnosing and treating cancer WO2010068850A1 (en)

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