WO2014015118A1 - Methods and compositions for the diagnosis and treatment of cellular proliferative disorders - Google Patents

Description

METHODS AND COMPOSITIONS FOR THE DIAGNOSIS AND TREATMENT OF CELLULAR PROLIFERATIVE DISORDERS

Cross-Reference to Related Applications

This application claims benefit of U.S. Provisional Application No. 61/674,000, filed July 20, 2012, which is hereby incorporated by reference in its entirety. Background of the Invention

In general, the invention relates to methods and compositions for the diagnosis and treatment of cellular proliferative disorders.

Cancer is caused in part by irreversible changes in DNA copy number at distinct locations in the genome. Aberrations of this type affect the function of genes and thereby produce a transformed phenotype, contributing to the manifestation and progression of cancer.

Particular aberrations may be distinct to particular cancer types. These findings empower diagnostic analysis on the basis of DNA copy number. Changes observed recurrently amongst tumors, or amongst tumors of a particular type, may represent functionally significant events.

Identification of functional copy number variants, and identification of critical genes within such regions once identified, is difficult. When such critical genes are identified, new avenues of treatment may be explored accordingly.

The altered metabolism of cancer cells is a functional change that provides an attractive target for therapeutic intervention. Cancer cells rely primarily on glycolysis for glucose metabolism. This phenomenon of altered metabolism in cancer cells, known as the Warburg effect, is characterized by increased glycolysis and decreased oxidative phosphorylation. Cancer cells demonstrate increased metabolism of glucose and glutamine, as well as the upregulation of enzymes and cofactors that detoxify reactive oxygen species (ROS).

There exists a need in the art for methods and compositions for diagnosing and treating cellular proliferative disorders. A gene with altered copy number in cancer cells that modulates metabolism and ROS detoxification would be of particular interest. Summary of the Invention

In one aspect, the invention features the use of a nicotinamide nucleotide

iranshvdrogenase (NNT) gene copy number in a biological sample in a method for diagnosing a cellular proliferative disorder (e.g., cancer) in a subject or assigning a prognostic risk of developing a cellular proliferative disorder in a subject, by determining a nicotinamide nucleotide transhydrogenase (NNT) gene copy number in a biological sample from the subject. In this method, an amplification (e.g., by 3-fold) of the NNT gene in the biological sample from the subject relative to a control gene copy number indicates the presence of a cellular proliferative disorder in the subject or the risk of developing the cellular proliferative disorder in the subject.

In another aspect, the invention features a method for diagnosing a cellular proliferative disorder in a subject (e.g., cancer) or assigning a prognostic risk of developing a cellular proliferative disorder (e.g., cancer) in a subject, the method including determining a NNT gene copy number in a biological sample from the subject. In this method, an amplification of the NNT gene in the biological sample (e.g., by at least 3 fold) from the subject relative to a control gene copy number indicates the presence of a cellular proliferative disorder in the subject or the risk of developing the cellular proliferative disorder in the subject.

In any of the methods or uses of the invention, NNT gene copy number can be determined by a hybridization- as say and/or an amplification-based assay, in situ hybridization (FISH), comparative genomic hybridization (CGH), or microarray-based CGH.

In another aspect, the invention features a method of identifying an inhibitor of nicotinamide nucleotide transhydrogenase (NNT), the method including:

(a) providing a cancer cell that expresses NNT or a functional fragment thereof.

(b) contacting a the cell with a candidate compound; and

(c) determining a level of NADPH present in the cell contacted with the candidate compound, where a reduction in the level of NADPH in the cell contacted with the candidate compound compared to a level of NADPH in a control cell not contacted with the candidate compound identifies the candidate compound as an inhibitor of NNT.

In a related aspect, the invention also features a method of identifying an inhibitor of nicotinamide nucleotide transhydrogenase (NNT), the method including:

(a) providing a cancer cell including NNT, or a functional fragment thereof, and NADP⁺.

(b) contacting the cancer cell with a candidate compound; and

(c) determining a level of NADPH present in the cancer cell, where a reduction in the level of NADPH in the sample contacted with the candidate compound compared to a level of NADPH in a control sample not contacted with the candidate compound identifies the candidate compound as an inhibitor of NNT.

In any of the above methods, the determining step can be performed using fluorescence spectroscopy.

In another aspect, the invention features a method of treating or reducing the likelihood of developing a cellular proliferative disorder (e.g., cancer) in a subject in need thereof (e.g., as characterized by an amplification of NNT gene or by a mutation or altered expression of K-ras) by administering to the subject a therapeutically effective amount of an inhibitor of nicotinamide nucleotide transhydrogenase (NNT).

In the methods of the invention, the inhibitor of NNT can reduce or inhibit the activity

(e.g., conversion of NADP⁺ to NADPH or the promotion of cell proliferation) or expression levels of an NNT polypeptide or nucleic acid molecule. The inhibitor of NNT can be a peptide, nucleic acid molecule (e.g., short interfering RNA (siRNA) or microRNA), aptamer, small molecule, or polysaccharide. An inhibitor can also be FSB A, NN'-dicyclohexylcarbodi-imide or butane-2,3-dione.

In another aspect, the invention features the use of an inhibitor of NNT for treating or reducing the likelihood of developing a cellular proliferative disorder (e.g., a disorder characterized by an amplification of an NNT gene) in a subject in need thereof by administering to the subject a therapeutically effective amount of an inhibitor of NNT.

In any of the methods or uses of the invention, the cancer can be prostate cancer, squamous cell cancer, small-cell lung cancer, non- small-cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma, gastric cancer, melanoma, or neck cancer.

The above methods and uses can further include administering to the subject an additional therapeutic agent (e.g., chemotherapeutic agent).

By "amplification" or "amplified" is meant the duplication, multiplication, or multiple expression of a gene or nucleic acid encoding a polypeptide, in vivo or in vitro, and refer to a process by which multiple copies of a gene or gene fragment are formed in a particular cell or cell line. The amount of messenger RNA (mRNA) produced, i.e., the level of gene expression, may also increase in proportion to the number of copies made of the particular gene. An NNT gene is said to be "amplified" if the genomic copy number of the NNT gene is higher than the control gene copy number, which is typically two copies per cell. In one example, an NNT gene is said to be "amplified" if the genomic copy number of the NNT gene is increased by at least 2-

(i.e., 6 copies), 3- (i.e., 8 copies), 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, or 50- fold in a test sample relative to a control sample. In another example, an NNT gene is said to be "amplified" if the genomic copy number of the NNT gene per cell is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and the like.

By "biological sample" or "sample" is meant solid and fluid samples. Biological samples may include cells, protein or membrane extracts of cells, tumors, or blood or biological fluids including, e.g., ascites fluid or brain fluid (e.g., cerebrospinal fluid (CSF)). Examples of solid biological samples include samples taken from feces, the rectum, central nervous system, bone, breast tissue, renal tissue, the uterine cervix, the endometrium, the head or neck, the gallbladder, parotid tissue, the prostate, the brain, the pituitary gland, kidney tissue, muscle, the esophagus, the stomach, the small intestine, the colon, the liver, the spleen, the pancreas, thyroid tissue, heart tissue, lung tissue, the bladder, adipose tissue, lymph node tissue, the uterus, ovarian tissue, adrenal tissue, testis tissue, the tonsils, and the thymus. Examples of biological fluid samples include samples taken from the blood, serum, CSF, semen, prostate fluid, seminal fluid, urine, saliva, sputum, mucus, bone marrow, lymph, and tears. Samples may be obtained by standard methods including, e.g., venous puncture and surgical biopsy. In certain embodiments, the biological sample is a breast, lung, colon, or prostate tissue sample obtained by needle biopsy.

By "cancer" and "cancerous" is meant the physiological condition in mammals that is typically characterized by abnormal cell growth. Included in this definition are benign and malignant cancers, as well as dormant tumors or micro-metastases. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particular examples of such cancers include, e.g., prostate cancer, squamous cell cancer, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma, gastric cancer, melanoma, and various types of head and neck cancer.

By "candidate compound" is meant a chemical, either naturally occurring or artificially derived. Candidate compounds may include, for example, peptides, polypeptides, synthetic organic molecules, naturally occurring organic molecules, nucleic acid molecules, peptide nucleic acid molecules, and components and derivatives thereof. Compounds useful in the invention include those described herein in any of their pharmaceutically acceptable forms, including isomers, such as diastereomers and enantiomers, salts, esters, solvates, and polymorphs thereof, as well as racemic mixtures and pure isomers of the compounds described herein.

By "cellular proliferation disorder" is meant a disorder associated with abnormal cell growth. Exemplary cell proliferative disorders include cancer (e.g., benign and malignant), obesity, benign prostatic hyperplasia, psoriasis, abnormal keratinization, lymphoproliferative disorders, rheumatoid arthritis, arteriosclerosis, restenosis, diabetic retinopathy, retrolental fibrioplasia, neovascular glaucoma, angiofibromas, hemangiomas, Karposi's sarcoma, and neurodegenerative disorders. Cellular proliferative disorders are described, for example, in U.S. Patent Nos. 5,639,600, 7,087,648, and 7,217,737, hereby incorporated by reference.

By "chemotherapeutic agent" is meant an agent that may be used to destroy a cancer cell or to slow, arrest, or reverse the growth of a cancer cell. Chemotherapeutic agents include, e.g., L-asparaginase, bleomycin, busulfan carmustine (BCNU), chlorambucil, cladribine (2-CdA), CPT11 (irinotecan), cyclophosphamide, cytarabine (Ara-C), dacarbazine, daunorubicin, dexamethasone, doxorubicin (adriamycin), etoposide, fludarabine, 5-fluorouracil (5FU), hydroxyurea, idarubicin, ifosfamide, interferon-a (native or recombinant), levamisole, lomustine (CCNU), mechlorethamine (nitrogen mustard), melphalan, mercaptopurine, methotrexate, mitomycin, mitoxantrone, paclitaxel, pentostatin, prednisone, procarbazine, tamoxifen, taxol- related compounds, 6-thiogaunine, topotecan, vinblastine, vincristine, cisplatinum,

carboplatinum, oxaliplatinum, or pemetrexed.

By "comparing" or "compared" is meant to include the act of providing, documenting, or memorializing data, information, or results relating to the same parameter from a test sample and a control sample. "Comparing" or "compared" also includes comparisons made indirectly.

By "control" or "control sample" is meant a biological sample representative or obtained from a healthy subject that has not been diagnosed with a cellular proliferative disorder. A control or control sample may have been previously established based on measurements from healthy subjects that have not been diagnosed with a cellular proliferative disorder. Further, a control sample can be defined by a specific age, sex, ethnicity, or other demographic parameters.

By "control gene copy number" of NNT is meant the gene copy number of the NNT gene in a control or control sample that is typical of the general population of healthy subjects that have not been diagnosed with a cellular proliferative disorder. In some embodiments, the control is implicit in the particular measurement. For example, a typical control level for a gene (i.e., control gene copy number) is two copies per cell. An example of an implicit control is where a detection method can only detect an NNT gene copy number when the copy number is higher than the typical control level. Other instances of such controls are within the knowledge of the skilled artisan. By "decrease" is meant to reduce by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,

75%, 80%, 85%, 90%, 95%, or more. A decrease can refer, for example, to the symptoms of the disorder being treated or to the levels or biological activity of a polypeptide or nucleic acid of the invention.

By "detection of expression" is meant the detection of a nucleic acid molecule or polypeptide by standard art known methods. For example, polypeptide expression is often detected by Western blotting, DNA expression is often detected by Southern blotting or polymerase chain reaction (PCR), and RNA expression is often detected by Northern blotting, PCR, or RNase protection assays.

By "functional fragment" is meant a portion of a polypeptide or nucleic acid molecule that contains at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the entire length of a nucleic acid molecule or polypeptide (e.g., NNT) that maintains biological activity. For example, a functional fragment of the NNT polypeptide may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or more amino acid residues, up to the full-length of the NNT.

By "increase" is meant to augment by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more. An increase can refer, for example, to the symptoms of the disorder being treated or to the levels or biological activity of a polypeptide or nucleic acid of the invention.

By "inhibitor" is meant any small molecule, nucleic acid molecule, peptide or

polypeptide, or fragments thereof that reduces or inhibits the expression levels or biological activity of a protein or nucleic acid molecule by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. Non-limiting examples of inhibitors include, e.g., small molecule inhibitors, antisense oligomers (e.g., morpholinos), double- stranded RNA for RNA interference (e.g., short interfering RNA (siRNA)), microRNA, aptamers, compounds that decrease the half- life of an mRNA or protein, compounds that decrease transcription or translation, dominant- negative fragments or mutant polypeptides that block the biological activity of wild-type protein, and peptidyl or non-peptidyl compounds (e.g., antibodies or antigen-binding fragments thereof) that bind to a protein.

By "pharmaceutical composition" is meant a composition containing a therapeutic agent of the invention (e.g., an inhibitor of NNT) formulated with a pharmaceutically acceptable excipient and manufactured for the treatment or prevention of a disorder in a subject.

Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gel-cap, or syrup), for topical administration (e.g., as a cream, gel, lotion, or ointment), for intravenous administration (e.g., as a sterile solution, free of particulate emboli, and in a solvent system suitable for intravenous use), or for any other formulation described herein.

By "pharmaceutically acceptable carrier" is meant a carrier that is physiologically acceptable to the treated subject while retaining the therapeutic properties of the therapeutic agent (e.g., an inhibitor of NNT) with which it is administered. One exemplary pharmaceutically acceptable carrier substance is physiological saline. Other physiologically acceptable carriers and their formulations are known to one skilled in the art.

By "pharmaceutically acceptable salt" is meant salts that are suitable for use in contact with the tissues of a subject without undue toxicity, irritation, or allergic response.

Pharmaceutically acceptable salts are well known in the art. The salts can be prepared in situ during the final isolation and purification of the therapeutic agents of the invention or separately by reacting the free base function with a suitable organic acid. Representative acid addition salts include, e.g., acetate, ascorbate, aspartate, benzoate, citrate, digluconate, fumarate,

glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, lactate, malate, maleate, malonate, mesylate, oxalate, phosphate, succinate, sulfate, tartrate, thiocyanate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine,

dimethylamine, trimethylamine, triethylamine, and ethylamine.

By "reduce or inhibit" is meant the ability to cause an overall decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater. For therapeutic

applications, to "reduce or inhibit" can refer to the symptoms of the disorder being treated or the presence or extent of a disorder being treated.

By "reducing the likelihood of is meant reducing the severity, the frequency, or both the severity and frequency of a cellular proliferative disorder or symptoms thereof. Reducing the likelihood of a cellular proliferative disorder is synonymous with prophylaxis or the chronic treatment of a cellular proliferative disorder.

By "reference" is meant any sample, standard, or level that is used for comparison purposes. A "normal reference sample" can be a prior sample taken from the same subject prior to the onset of a disorder (e.g., a cellular proliferation disorder), a sample from a subject not having the disorder, a subject that has been successfully treated for the disorder, or a sample of a purified reference polypeptide at a known normal concentration. By "reference standard or level" is meant a value or number derived from a reference sample. A normal reference standard or level can be a value or number derived from a normal subject that is matched to a sample of a subject by at least one of the following criteria: age, weight, disease stage, and overall health. A

"positive reference" sample, standard, or value is a sample, standard, value, or number derived from a subject that is known to have a disorder (e.g., a cellular proliferation disorder) that is matched to a sample of a subject by at least one of the following criteria: age, weight, disease stage, and overall health.

By "subject" is meant any animal, e.g., a mammal (e.g., a human). A subject who is being treated for, e.g., a cellular proliferative disorder (e.g., cancer and obesity) is one who has been diagnosed by a medical practitioner as having such a condition. Diagnosis may be performed by any suitable means. A subject of the invention may be one that has not yet been diagnosed with a cellular proliferative disorder. A subject of the invention may be identified as one having an amplification of the NNT gene. One of skill in the art will understand that subjects treated using the compositions or methods of the present invention may have been subjected to standard tests or may have been identified without examination as one at high risk due to the presence of one or more risk factors, such as age, genetics, or family history.

By "systemic administration" is meant any non-dermal route of administration and specifically excludes topical and transdermal routes of administration.

By "therapeutic agent" is meant any agent that produces a healing, curative, stabilizing, or ameliorative effect.

By "treating" is meant administering a pharmaceutical composition for prophylactic and/or therapeutic purposes. Prophylactic treatment may be administered, for example, to a subject who is not yet ill, but who is susceptible to, or otherwise at risk of, a particular disorder, e.g., a cellular proliferation disorder (e.g., cancer and obesity). Therapeutic treatment may be administered, for example, to a subject already suffering from a disorder in order to improve or stabilize the subject's condition. In some instances, as compared with an equivalent untreated control, treatment may ameliorate a disorder or a symptom thereof by, e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% as measured by any standard technique. In some instances, treating can result in the inhibition of a disease, the healing of an existing disease, and the amelioration of a disease.

Other features and advantages of the invention will be apparent from the following detailed description, the claims, and the drawings.

Brief Description of Drawings

Fig L is a Tumorscape genome browser view of an NNT amplification peak containing six genes (NNT, C50RF28, C50RF34, FGF10, MRPS30 and PAIP1) of Chromosome 5p. Fig 2. is a graph showing NNT mRNA expression in NSCLC cells. The graph shows

NNT- amplified NSCLC cell lines (left column) and non-amplified cell lines (right column).

Fig 3. is a pair of immunohistochemistry images showing NNT protein expression in representative normal lung (top image) and NSCLC tumor (bottom image) cells. In both images, nuclei are stained in gray by hematoxylin and NNT protein is stained in dark gray.

Fig 4. is a series of images of Western blot data showing NNT protein expression in NNT-amplifed and non-amplified NSCLC cell lines. Expression of Actin protein is provided as a control.

Fig. 5 is an image of a western blot showing the expression of NNT and actin in H2009 cancer cells infected with lentiviral vectors encoding either one of five tested shNNT constructs (shNNT 28489, shNNT 28507, shNNT 28512, shNNT 28513, or shNNT 28541) or a scrambled hairpin sequence (scr) control.

Fig. 6 is an image of a western blot showing the expression of NNT and actin in three cancer cell types (PC9, H2009, and H1299) transfected with siNNT or an siRNA that does not target NNT.

Fig. 7 is a series of three graphs showing the analysis of intracellular NADPH, ROS, and caspase activation in PC9 cells treated with NNT shRNA. Fig. 7A shows the NADP+/NADPH ratio in PC9 cells three days after infection with lentiviruses encoding scrambled shRNA or shRNA targeting NNT. Fig. 7B shows the intracellular level of ROS as measured using dichlorohydrofluorescein diacetate (DCF) in PC9 cells after infection with lentiviruses encoding scrambled shRNA or shRNA targeting NNT. Fig. 7C shows caspase activation in PC9 cells as measured by Caspase 3/7 Glo (Promega) 4 days after infection with lentiviruses encoding scrambled shRNA or shRNA targeting NNT.

Fig. 8 is a series of four graphs showing the growth of cells following treatment with a scrambled shRNA control (datapoints shown as diamonds) or NNT knock-down with shRNA (datapoints showin as triangles) in four non-amplified NSCLC lines.

Fig. 9 is a pair of graphs showing the growth of cells following treatment with a scrambled shRNA control (datapoints shown as diamonds) or NNT knock-down with shRNA (datapoints shown as triangles) in two NNT- amplified NSCLC lines (H2009 and PC9).

Detailed Description of the Invention

In general, the invention features compositions and methods for treating cellular proliferative disorders. This invention is based on the discovery that nicotinamide nucleotide transhydrogenase (NNT) is amplified in cancer tissue (e.g., lung cancer tissue), that NNT is overexpressed at the protein and mRNA level in non- small cell lung carcinoma cells (NSCLC cells), and that inhibition of NNT both induces cell death in NNT-amplified NSCLC cell lines and inhibits proliferation of non-amplified cell lines. Accordingly, the invention features methods of diagnosing cancer (e.g., NSCLC) by determining whether the NNT gene is amplified, or if NNT is overexpressed, in a sample (e.g., a tumor or blood sample). Furthermore, the invention features methods of treating cancer (e.g., NSCLC) using inhibitors of NNT (e.g., RNAi compounds, antagonistic antibodies, or small molecule inhibitors). Such methods of treatment can be performed in cancer patients in general, or in patients found to have an NNT gene amplification or increased expression of NNT. Finally, the invention features methods of identifying compounds useful for the treatment of cancer (e.g., NSCLC) by screening for compounds that inhibit NNT activity (e.g., NNT activity in cancer cells and/or cells with an NNT gene amplification or NNT overexpression).

Genetic lesions are characteristic of cells presenting proliferative diseases, and a subset of these lesions contribute to the disease. These lesions may involve a change in copy number of genes or groups of genes. NNT is amplified in nearly 1/3 (30.34%) of all cancers and 57.71 of NSC lung cancers.

NNT is an important regulator of the mitochondrial redox state. It is an integral protein of the inner mitochondrial membrane, and contributes significantly to mitochondrial NADPH. NNT is a proton-translocationg transhydrogenase that utilizes energy from the mitochondrial proton gradient to catalyze the conversion of NADH to NADPH. The NADPH generated by NNT can be used for biosynthesis reactions and to detoxify reactive oxygen species (ROS) generated as a byproduct of mitochondrial metabolism. Excessive ROS generation results in damage to the mitochondria and the cell, and eventually death. Therefore the activity of

NADPH-generating enzymes is critical to overall cellular health and growth. Our research demonstrates that amplification of NNT is functionally significant in the progress of proliferative disease. We find that inhibition of NNT inhibits the progress of proliferative disease. These findings provide a direct link between a previously unknown genetic lesion particular to proliferative disease and cellular metabolism, enabling novel modes of diagnosis and treatment for proliferative diseases. Cellular Proliferative Disorders

The present invention features methods and compositions for the diagnosis and prognosis of cellular proliferative disorders (e.g., cancer) and the treatment of these disorders by targeting NNT. Cellular proliferative disorders described herein include, e.g., cancer, obesity, and proliferation-dependent diseases. Such disorders may be diagnosed using methods known in the art. Cancer

Cancers include, without limitation, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease or non-Hodgkin's disease), Waldenstrom's

macro globulinemia, multiple myeloma, heavy chain disease, 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, bile 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, oligodendroglioma,, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma), prostate cancer, squamous cell cancer, small-cell lung cancer, non-small-cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma, gastric cancer, melanoma, or neck cancer.

Other Proliferative Diseases

Other proliferative diseases include, e.g., obesity, benign prostatic hyperplasia, psoriasis, abnormal keratinization, lymphoproliferative disorders (e.g., a disorder in which there is abnormal proliferation of cells of the lymphatic system), chronic rheumatoid arthritis, arteriosclerosis, restenosis, and diabetic retinopathy. Proliferative diseases are described in U.S. Patent Nos. 5,639,600 and 7,087,648, hereby incorporated by reference. Diagnostics

The present invention features methods to diagnose a cellular proliferative disorder and monitor the progression of such a disorder. For example, the methods can include determining NNT gene copy number in a biological sample and comparing the gene copy number to a normal reference.

Determination of the genomic copy number of NNT has many advantages over determining, for example, the protein level or mRNA expression level of NNT in a cell. Many cells, including non-cancer cells, express NNT. However, expression at the protein or mRNA level alone may not be sufficient to identify those cancers which were selected specifically to have a genetic event leading to increased NNT expression. In contrast, amplification of the gene suggests a genetic selection for those cells which are dependent on higher copy number of NNT for growth. In these cells, NNT expression provides a growth advantage that enables the clonal expansion of cells with the genomic alteration leading to increased expression. Thus, examination of the genomic copy number can identify those cancers which will respond to therapy targeting NNT.

The presence of a gene that has undergone amplification in a biological sample is evaluated by determining the copy number of the genes, e.g., the number of DNA sequences in a cell encoding the target protein. Generally, a normal diploid cell has two copies of a given autosomal gene. The copy number can be increased, however, by gene amplification or duplication, for example, in cancer cells, or reduced by deletion. Methods of evaluating the copy number of a particular gene are well known in the art and include, without limitation, hybridization- and amplification-based assays.

Any of a number of hybridization-based assays can be used to detect the copy number of, for example, an NNT gene in a biological sample. One such method is Southern blotting, where the genomic DNA may be fragmented, separated electrophoretically, transferred to a membrane, and subsequently hybridized to an NNT-specific probe. Comparison of the intensity of the hybridization signal from the probe for the target region with a signal from a control probe from a region of normal non-amplified, single-copied genomic DNA in the same genome provides an estimate of the relative NNT gene copy number, corresponding to the specific probe used. An increased signal compared to a control represents the presence of amplification.

Another methodology for determining the copy number of the NNT gene in a sample is in situ hybridization, for example, fluorescence in situ hybridization (FISH) (see, e.g., Angerer et al., Methods Enzymol. 152:649-661, 1987). Generally, in situ hybridization includes the following steps: (1) fixation of a biological sample to be analyzed; (2) pre-hybridization treatment of the biological sample to increase accessibility of target DNA and to reduce nonspecific binding; (3) hybridization of the mixture of nucleic acids to the nucleic acid in the biological sample; (4) post-hybridization washes to remove nucleic acid fragments not bound in the hybridization; and (5) detection of the hybridized nucleic acid fragments. The probes used in such applications are typically labeled, for example, with radioisotopes or fluorescent reporters. Preferred probes are sufficiently long, for example, from about 50, 100, or 200 nucleotides to about 1000 or more nucleotides, to enable specific hybridization with the target nucleic acid(s) under stringent conditions.

Another methodology for determining the number of gene copies is comparative genomic hybridization (CGH). In comparative genomic hybridization methods, a "test" collection of nucleic acids is labeled with a first label, while a second collection (for example, from a normal cell or tissue) is labeled with a second label. The ratio of hybridization of the nucleic acids is determined by the ratio of the first and second labels binding to each fiber in an array.

Differences in the ratio of the signals from the two labels, for example, due to gene amplification in the test collection are detected, and the ratio provides a measure of, for example, the gene copy number corresponding to the specific probe used. A cytogenetic representation of DNA copy-number variation can be generated by CGH, which provides fluorescence ratios along the length of chromosomes from differentially labeled test and reference genomic DNAs.

Hybridization protocols suitable for use with the methods of the invention are described, for example, in Albertson, EMBO J. 3: 1227-1234, 1984, and Pinkel et al., Proc. Natl. Acad. Sci. USA 85:9138-9142, 1988, hereby incorporated by reference.

Amplification-based assays also can be used to measure the copy number of the NNT gene. In such assays, the corresponding NNT nucleic acid sequences act as a template in an amplification reaction (for example, a polymerase chain reaction or PCR). In a quantitative amplification, the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the copy number of the NNT gene, corresponding to the specific probe used, according to the principles discussed above. Methods of real-time quantitative PCR using TaqMan probes are well known in the art. Detailed protocols for real-time quantitative PCR are provided, for example, in Gibson et al., Genome Res. 6:995-1001, 1996, and in Heid et al., Genome Res. 6:986-994, 1996.

A TaqMan-based assay also can be used to quantify NNT polynucleotides. TaqMan- based assays use a fluorogenic oligonucleotide probe that contains a 5' fluorescent dye and a 3' quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3' end. When the PCR product is amplified in subsequent cycles, the 5' nuclease activity of the polymerase, for example, AmpliTaq, results in the cleavage of the TaqMan probe. This cleavage separates the 5' fluorescent dye and the 3' quenching agent, thereby resulting in an increase in fluorescence as a function of amplification.

Other suitable amplification methods include, but are not limited to, ligase chain reaction (LCR) (see, e.g., Wu and Wallace, Genomics 4:560-569, 1989; Landegren et al., Science 241: 1077-1080, 1988; and Barringer et al., Gene 89: 117-122, 1990), transcription amplification (see, e.g., Kwoh et al., Proc. Natl. Acad. Sci. USA 86: 1173-1177, 1989), self-sustained sequence replication (see, e.g., Guatelli et al., Proc. Natl. Acad. Sci. USA 87: 1874-1878, 1990), dot PCR, and linker adapter PCR.

DNA copy number may also be determined using microarray-based platforms (e.g., single-nucleotide polymorphism (SNP) arrays), as microarray technology offers high resolution. For example, traditional CGH generally has a 20 Mb-limited mapping resolution, whereas, in microarray-based CGH, the fluorescence ratios of the differentially labeled test and reference genomic DNAs provide a locus-by-locus measure of DNA copy-number variation, thereby achieving increased mapping resolution. Details of various microarray methods can be found in the literature. See, for example, U.S. Patent No. 6,232,068 and Pollack et al., Nat. Genet. 23:41- 46, 1999.

Detection of amplification, overexpression, or overproduction of, for example, an NNT gene or gene product can also be used to provide prognostic information or guide therapeutic treatment. Such prognostic or predictive assays can be used to determine prophylactic treatment of a subject prior to the onset of symptoms of, e.g., a cellular proliferative disorder.

The methods of the present invention can also include the detection and measurement of, for example, NNT (or a functional fragment thereof) expression or biological activity.

For diagnoses based on relative levels of NNT, a subject with a disorder (e.g., a cellular proliferative disorder) will show an alteration (e.g., an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) in the amount of the NNT expressed or an alteration (e.g., an increase of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more) in NNT biological activity compared to a normal reference. A normal reference sample can be, for example, a prior sample taken from the same subject prior to the development of the disorder or of symptoms suggestive of the disorder, a sample from a subject not having the disorder, a sample from a subject not having symptoms of the disorder, or a sample of a purified reference polypeptide at a known normal concentration (i.e., not indicative of the disorder).

Standard methods may be used to measure levels of NNT in a biological sample, including, but not limited to, tumor, urine, blood, serum, plasma, saliva, amniotic fluid, or cerebrospinal fluid. Such methods include immunoassay, ELISA, Western blotting, and quantitative enzyme immunoassay techniques, such as IHC. The diagnostic methods described herein can be used individually or in combination with any other diagnostic method described herein for a more accurate diagnosis of the presence or severity of a disorder (e.g., a cellular proliferation disorder). Examples of additional methods for diagnosing such disorders include, e.g., examining a subject's health history,

immunohistochemical staining of tissues, computed tomography (CT) scans, culture growths, or determining if the subject has altered K-ras expression or a mutation in K-ras.

Screening Assays

Inhibiting NNT inhibits cell proliferation. NNT is therefore a useful targets for high- throughput, low-cost screening of candidate compounds to identify those that modulate, alter, or decrease (e.g., by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) the expression or biological activity of NNT. Compounds that decrease the expression or biological activity of NNT can be used for the treatment of a cellular proliferative disorder. Candidate compounds can be tested for their effect on NNT using assays known in the art or described in the Examples below.

For example, NNT contributes to the production of NADPH such that inhibition NNT inhibits the production of NADPH. Accordingly, to identify inhibitors of NNT, conversion of NADP⁺ to NADPH can be monitored (e.g., in vitro or in vivo) when NNT is contacted with a candidate compound. A decrease in the conversion of NADP⁺ to NADPH may indicate, for example, that the candidate compound is an inhibitor of NNT. The conversion of NADP⁺ to NADPH can be monitored directly or indirectly, for example, using diaphorase as a detection enzyme system or any other methods known in the art. The conversion of NADP⁺ to NADPH can also monitored through monitoring the consumption of NADP⁺ or the production of

NADPH. The consumption of NADP⁺ or the production of NADPH can be monitored directly or indirectly.

Methods of identifying NNT inhibitors are known in the art, such as those described in Meadows et al., J Biomol Screen 16: 734-743; Yamaguchi and Hatefi, The Journal of Biological Chemistry, 270, 47, Nov. 24: 28165-28168; Yamaguchi and Hatefi, Biochemica et Biophysica Acta 1318: 225-234; Yin et al., Biochemica et Biophysica Acta 1817:401-409; Rydstrom

Methods in enzymology, 1979. 55: p. 261-75; Peake et al. Biochimica et Biophysica Acta. 1999 1411: p. 159-169; and Moody and Reid, Biochem J 209: 889-892, which are hereby

incorporated by reference.

In general, candidate compounds are identified from large libraries of natural product or synthetic (or semi-synthetic) extracts, chemical libraries, or from polypeptide or nucleic acid libraries, according to methods known in the art. Those skilled in the field of drug discovery and development will understand that the precise source of test extracts or compounds is not critical to the screening procedure(s) of the invention.

Therapeutic Agents

Therapeutic agents useful in the methods of the invention include any compound that can reduce or inhibit the biological activity or expression level of NNT. Exemplary inhibitor compounds include, but are not limited to, small molecule inhibitors, antisense nucleobase oligomers (e.g., morpholinos), double- stranded RNA for RNA interference (e.g., short interfering RNA (siRNA)), microRNA, aptamers, compounds that decrease the half-life of an mRNA or protein, compounds that decrease transcription or translation, dominant-negative fragments or mutant polypeptides that block the biological activity of wild-type protein, and peptidyl or non-peptidyl compounds (e.g., antibodies or antigen-binding fragments thereof) that bind to a protein (e.g., NNT).

Desirably, inhibitor compounds will reduce or inhibit the biological activity or expression levels of polypeptide or nucleic acid (e.g., an NNT polypeptide or nucleic acid) by at least 10%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more. The inhibitor compound may reduce or inhibit cell proliferation or the reduction of NADP⁺ to NAPDH by at least 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or more.

Nucleic Acid Molecules

The therapeutic agent of the invention (e.g., an inhibitor of NNT) may be a nucleic acid molecule. Such inhibitory nucleic acid molecules are capable of mediating the downregulation of the expression of a polypeptide or nucleic acid encoding the same (e.g., an NNT polypeptide or nucleic acid) or mediating a decrease in the activity of a polypeptide of the invention.

Examples of the inhibitory nucleic acids of the invention include, without limitation, antisense oligomers (e.g., morpholinos), dsRNAs (e.g., siRNAs and shRNAs), microRNAs, and aptamers.

Antisense Oligomers

The present invention features antisense oligomers to any of the polypeptides of NNT and the use of such oligomers to downregulate expression of mRNA encoding the polypeptide. By binding to the complementary nucleic acid sequence (i.e., the sense or coding strand), antisense oligomers are able to inhibit protein expression, presumably through the enzymatic cleavage of the RNA strand by RNase H. Desirably, the antisense oligomer is capable of reducing polypeptide expression in a cell by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater, relative to cells treated with a control oligonucleotide. Methods for selecting and preparing antisense oligomers are well known in the art. Methods for assaying levels of protein expression are also well known in the art and include, for example, Western blotting, immunoprecipitation, and ELISA.

One example of an antisense oligomer is a morpholino oligomer. Morpholinos act by "steric blocking" or binding to a target sequence within an RNA and blocking molecules, which might otherwise interact with the RNA.

Morpholinos are synthetic molecules that bind to complementary sequences of RNA by standard nucleic acid base-pairing. While morpholinos have standard nucleic acid bases, those bases are bound to morpholine rings instead of deoxyribose rings and linked through

phosphorodiamidate groups instead of phosphates. Because of their unnatural backbones, morpholinos are not recognized by cellular proteins. Nucleases do not degrade morpholinos, and morpholinos do not activate innate immune responses. Morpholinos are also not known to modify methylation of DNA. Accordingly, morpholinos that are directed to any part of a polypeptide of NNT and that reduce or inhibit the expression levels or biological activity of the polypeptide are particularly useful in the methods and compositions of the invention. dsRNAs

The present invention also features the use of double stranded RNAs including, but not limited to, siRNAs and shRNAs. Short, double-stranded RNAs may be used to perform RNA interference (RNAi) to inhibit the expression of a polypeptide of NNT. RNAi is a form of post- transcriptional gene silencing initiated by the introduction of double- stranded RNA (dsRNA). Short 15 to 32 nucleotide double-stranded RNAs, known generally as "siRNAs," "small RNAs," or "microRNAs" are effective at down-regulating gene expression in nematodes (Zamore et al., Cell 101: 25-33) and in mammalian tissue culture cell lines (Elbashir et al., Nature 411:494-498, 2001). The further therapeutic effectiveness of this approach in mammals was demonstrated in vivo by McCaffrey et al. (Nature 418: 38-39, 2002). The small RNAs are at least 15 nucleotides, preferably 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 nucleotides in length and even up to 50 or 100 nucleotides in length (inclusive of all integers in between). Such small RNAs that are substantially identical to or complementary to any region of a polypeptide described herein are included in the invention. Non-limiting examples of small RNAs are substantially identical to (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity) or complementary to the NNT nucleic acid sequence. It should be noted that longer dsRNA fragments that are processed into small RNAs may be used. Small RNAs to be used as inhibitors of the invention can be identified by their ability to decrease polypeptide expression levels or biological activity performing assays known in the art or provided herein. Small RNAs can also include short hairpin RNAs in which both strands of an siRNA duplex are included within a single RNA molecule.

The specific requirements and modifications of small RNAs are known in the art and are described, for example, in PCT Publication No. WO 01/75164, and U.S. Patent Application Publication Nos. 2006/0134787, 2005/0153918, 2005/0058982, 2005/0037988, and

2004/0203145, the relevant portions of which are herein incorporated by reference.

siRNA molecules can be obtained and purified through a variety of protocols known to one of skill in the art, including chemical synthesis or recombinant production using a

Drosophila in vitro system. They are commercially available from companies such as

Dharmacon Research Inc. or Xeragon Inc., or they can be synthesized using commercially available kits such as the Silencer™ siRNA Construction Kit from Ambion (Catalog Number 1620) or HiScribe™ RNAi Transcription Kit from New England BioLabs (Catalog Number E2000S). Alternatively, siRNA can be prepared using standard procedures for in vitro transcription of RNA and dsRNA annealing procedures.

Short hairpin RNAs (shRNAs) can also be used in the methods of the invention. shRNAs are designed such that both the sense and antisense strands are included within a single RNA molecule and connected by a loop of nucleotides. shRNAs can be synthesized and purified using standard in vitro T7 transcription synthesis. shRNAs can also be subcloned into an expression vector, which can then be transfected into cells and used for in vivo expression of the shRNA.

A variety of methods are available for transfection of dsRNA into mammalian cells. For example, there are several commercially available transfection reagents useful for lipid-based transfection of siRNAs including, but not limited to, TransIT-TKO™ (Minis, Catalog Number MIR 2150), Transmessenger™ (Qiagen, Catalog Number 301525), Oligofectamine™ and Lipofectamine™ (Invitrogen, Catalog Number MIR 12252-011 and Catalog Number 13778- 075), siPORT™ (Ambion, Catalog Number 1631), DharmaFECT™ (Fisher Scientific, Catalog Number T-2001-01). Agents are also commercially available for electroporation-based methods for transfection of siRNA, such as siPORTer™ (Ambion Inc., Catalog Number 1629).

Microinjection techniques may also be used. The small RNA can also be transcribed from an expression construct introduced into the cells, where the expression construct includes a coding sequence for transcribing the small RNA operably linked to one or more transcriptional regulatory sequences. Where desired, plasmids, vectors, or viral vectors can also be used for the delivery of dsRNA or siRNA, and such vectors are known in the art. Protocols for each transfection reagent are available from the manufacturer. Additional methods are known in the art and are described, for example, in U.S. Patent Application Publication No. 2006/0058255. Aptamers

The present invention also features aptamers to the polypeptides of the invention (e.g., NNT) and the use of such aptamers to downregulate expression of the polypeptide or nucleic acid encoding the polypeptide. Aptamers are nucleic acid molecules that form tertiary structures that specifically bind to a target molecule. The generation and therapeutic use of aptamers are well established in the art. See, e.g., U.S. Patent No. 5,475,096 and U.S. Patent Application Publication No. 2006/0148748. For example, an NNT aptamer may be a pegylated, modified oligonucleotide, which adopts a three-dimensional conformation that enables it to bind to NNT and inhibit the biological activity of NNT.

Small Molecule Therapeutic Agents

Small molecule therapeutic agents for use in the present invention can be identified using standard screening methods specific to NNT. These screening methods can also be used to confirm the activities of derivatives of compounds found to have a desired activity, which are designed according to standard medicinal chemistry approaches. After a small molecule therapeutic agent is confirmed as being active with respect to a particular target, the therapeutic agent can be tested in vitro, as well as in appropriate animal model systems.

The small molecule therapeutic agents of the present invention may be derivatives, analogs, or mimetics of known NNT inhibitors, including but not limited to NN'- dicyclohexylcarbodi-imide (DCCD), butane-2,3-dione (butanedione), diethylpyrocarbonate, N- (ethoxycarbonyl)-2-ethoxy-l,2-dihydroquinoline (EEDQ), or 5'-[p-(fluorosulfonyl)benzoyl]- adenosine (FSBA). Therapeutic Formulations

The invention includes the use of therapeutic agents (e.g., inhibitor compounds) to treat or reduce the likelihood of developing a cellular proliferative disorder (e.g., cancer and obesity) in a subject. Thus, the present invention includes pharmaceutical compositions that include an inhibitor of NNT and a pharmaceutically acceptable carrier, wherein said inhibitor of NNT is present in an amount that, when administered to a subject, is sufficient to treat or reduce the likelihood of developing a cellular proliferative disorder in said subject. In one aspect, the cellular proliferative disorder is cancer. The therapeutic agent can be administered at any time. For example, for therapeutic applications, the agent can be administered after diagnosis or detection of a cellular proliferative disorder or after the onset of symptoms of a cellular proliferative disorder. The therapeutic agent can also be administered before diagnosis or onset of symptoms of a cellular proliferative disorder in subjects that have not yet been diagnosed with a cellular proliferative disorder, but that are at risk of developing such a disorder, or after a risk of developing a cellular proliferative disorder is determined. A therapeutic agent of the invention may be formulated with a pharmaceutically acceptable diluent, carrier, or excipient in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the therapeutic agent of the invention to a subject suffering from or at risk of developing a cellular proliferative disorder. Administration may begin before the patient is symptomatic. The therapeutic agent of the present invention can be formulated and administered in a variety of ways, e.g., those routes known for specific indications, including, but not limited to, topically, orally, subcutaneously, intravenously, intracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, rectally, intra- arterially, intralesionally, parenterally, or intra-ocularly. The therapeutic agent can be in the form of a pill, tablet, capsule, liquid, or sustained release tablet for oral administration; or a liquid for intravenous administration, subcutaneous administration, or injection; for intranasal formulations, in the form of powders, nasal drops, or aerosols; or a polymer or other sustained-release vehicle for local administration.

The invention also includes the use of therapeutic agent (e.g., an inhibitor of NNT) to treat or reduce the likelihood of developing a cellular proliferative disorder in a biological sample derived from a subject (e.g., treatment of a biological sample ex vivo) using any means of administration and formulation described herein). The biological sample to be treated ex vivo may include any biological fluid (e.g., blood, serum, plasma, or cerebrospinal fluid), cell (e.g., an endothelial cell), or tissue from a subject that has a cellular proliferative disorder or the propensity to develop a cellular proliferative disorder. The biological sample treated ex vivo with the therapeutic agent may be reintroduced back into the original subject or into a different subject. The ex vivo treatment of a biological sample with a therapeutic agent, as described herein, may be repeated in an individual subject (e.g., at least once, twice, three times, four times, or at least ten times). Additionally, ex vivo treatment of a biological sample derived from a subject with a therapeutic agent, as described herein, may be repeated at regular intervals (non- limiting examples include daily, weekly, monthly, twice a month, three times a month, four times a month, bi-monthly, once a year, twice a year, three times a year, four times a year, five times a year, six times a year, seven times a year, eight times a year, nine times a year, ten times a year, eleven times a year, and twelve times a year).

Therapeutic formulations are prepared using standard methods known in the art by mixing the active ingredient having the desired degree of purity with optional physiologically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (20th edition), ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, PA) in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, include saline, or buffers such as phosphate, citrate and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEEN™, PLURONICS™, or PEG.

Optionally, the formulation contains a pharmaceutically acceptable salt (e.g., sodium chloride) at about physiological concentrations. The formulation may also contain the therapeutic agent (e.g., inhibitor of NNT) in the form of a calcium salt. The formulations of the invention may contain a pharmaceutically acceptable preservative. In some embodiments, the preservative concentration ranges from 0.1 to 2.0%, typically v/v. Suitable preservatives include those known in the pharmaceutical arts, including benzyl alcohol, phenol, m-cresol,

methylparaben, and propylparaben. The formulations of the invention may also include a pharmaceutically acceptable surfactant, such as non-ionic detergents.

For parenteral administration, the therapeutic compound is formulated in a unit dosage injectable form (e.g., solution, suspension, emulsion) in association with a pharmaceutically acceptable parenteral vehicle. Such vehicles are inherently non-toxic and non-therapeutic.

Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate may also be used.

Liposomes may be used as carriers. The vehicle may contain minor amounts of additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives.

The dosage required depends on the choice of the route of administration; the nature of the formulation; the nature of the subject's illness; the subject's size, weight, surface area, age, and sex; other drugs being administered; and the judgment of the attending physician. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art. Administrations can be single or multiple (e.g., 2, 3, 6, 8, 10, 20, 50, 100, 150, or more). Encapsulation of the therapeutic compound in a suitable delivery vehicle (e.g., polymeric microparticles or implantable devices) may increase the efficiency of delivery, particularly for oral delivery.

As described above, the dosage of the therapeutic agent will depend on other clinical factors such as weight and condition of the subject and the route of administration of the compound. For treating subjects, between approximately 0.001 mg/kg to 500 mg/kg body weight of the therapeutic agent (e.g., inhibitor of NNT) can be administered. A more preferable range is 0.01 mg/kg to 50 mg/kg body weight with the most preferable range being from 1 mg/kg to 25 mg/kg body weight. Depending upon the half-life of the therapeutic agent in the particular subject, the compound can be administered between several times per day to once a week. The methods of the present invention provide for single as well as multiple administrations, given either simultaneously or over an extended period of time.

Alternatively, a polynucleotide containing a nucleic acid sequence which is itself or encodes a therapeutic agent (e.g., an inhibitory nucleic acid molecule that inhibits the expression of a nucleic acid molecule encoding a polypeptide of NNT) can be delivered to the appropriate cells in the subject. Expression of the coding sequence can be directed to any cell in the body of the subject, preferably a cancer cell or adipocyte. This can be achieved, for example, through the use of polymeric, biodegradable microparticle or microcapsule delivery devices known in the art.

The nucleic acid can be introduced into the cells by any means appropriate for the vector employed. Many such methods are well known in the art. Examples of methods of gene delivery include, for example, liposome-mediated transfection, electroporation, calcium phosphate/DEAE dextran methods, gene gun, and microinjection. Delivery of "naked DNA" (i.e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site is another means to achieve in vivo expression. Gene delivery using viral vectors such as adenoviral, retroviral, lentiviral, or adeno-asociated viral vectors can also be used. An ex vivo strategy can also be used for therapeutic applications, as described herein. Ex vivo strategies involve transfecting or transducing cells obtained from the subject with a therapeutic nucleic acid compound. The transfected or transduced cells are then returned to the subject. Such cells act as a source of the therapeutic nucleic acid compound for as long as they survive in the subject.

The therapeutic agent can be packaged alone or in combination with other therapeutic agents as a kit. Additional therapeutic agents that can be used in combination with the therapeutic agents of the invention include chemo therapeutic agents. The kit can include optional components that aid in the administration of the unit dose to subjects, such as vials for reconstituting powder forms, syringes for injection, customized IV delivery systems, or inhalers. Additionally, the unit dose kit can contain instructions for preparation and administration of the compositions. The kit may be manufactured as a single use unit dose for one subject, multiple uses for a particular subject (e.g., at a constant dose or in which the individual compounds may vary in potency as therapy progresses), or the kit may contain multiple doses suitable for administration to multiple subjects (e.g., "bulk packaging"). The kit components may be assembled in cartons, blister packs, bottles, or tubes. Combination Therapies

Therapeutic compounds that inhibit NNT can be used alone or in combination with one, two, three, four, or more of the therapeutic agents of the invention or with a known therapeutic agent for the treatment or prevention of a cellular proliferative disorder, such as a

chemotherapeutic agent. Chemotherapeutic agents include, e.g., alkylating agents (e.g., busulfan, dacarbazine, ifosfamide, hexamethylmelamine, thiotepa, dacarbazine, lomustine, cyclophosphamide chlorambucil, procarbazine, altretamine, estramustine phosphate,

mechlorethamine, streptozocin, temozolomide, and Semustine), platinum agents (e.g., spiroplatin, tetraplatin, ormaplatin, iproplatin, ZD-0473 (AnorMED), oxaliplatin, carboplatin, lobaplatin (Aeterna), satraplatin (Johnson Matthey), BBR-3464 (Hoffmann-La Roche), SM- 11355 (Sumitomo), AP-5280 (Access), and cisplatin), antimetabolites (e.g., azacytidine, floxuridine, 2-chlorodeoxyadenosine, 6-mercaptopurine, 6-thioguanine, cytarabine, 2- fluorodeoxy cytidine, methotrexate, tomudex , fludarabine, raltitrexed, trimetrexate,

deoxycoformycin, pentostatin, hydroxyurea, decitabine (SuperGen), clofarabine (Bioenvision), irofulven (MGI Pharma), DMDC (Hoffmann-La Roche), ethynylcytidine (Taiho), gemcitabine, and capecitabine), topoisomerase inhibitors (e.g., amsacrine, epirubicin, etoposide, teniposide or mitoxantrone, 7-ethyl-lO-hydroxy-camptothecin, dexrazoxanet (TopoTarget), pixantrone (Novuspharma), rebeccamycin analogue (Exelixis), BBR-3576 (Novuspharma), rubitecan (SuperGen), irinotecan (CPT-11), topotecan, exatecan mesylate (Daiichi), quinamed

(ChemGenex), gimatecan (Sigma-Tau), diflomotecan (Beaufour-Ipsen), TAS-103 (Taiho), elsamitrucin (Spectrum), J-107088 (Merck & Co), BNP-1350 (BioNumerik), CKD-602 (Chong Kun Dang), KW-2170 (Kyowa Hakko), and hydroxycamptothecin (SN-38)), antitumor antibiotics (e.g., valrubicin, therarubicin, idarubicin, rubidazone, plicamycin, porfiromycin, mitoxantrone (novantrone), amonafide, azonafide, anthrapyrazole, oxantrazole, losoxantrone, MEN- 10755 (Menarini), GPX-100 (Gem Pharmaceuticals), epirubicin, mitoxantrone, and doxorubicin), antimitotic agents (e.g., colchicine, vinblastine, vindesine, dolastatin 10 (NCI), rhizoxin (Fujisawa), mivobulin (Warner-Lambert), cemadotin (BASF), RPR 109881 A (Aventis), TXD 258 (Aventis), epothilone B (Novartis), T 900607 (Tularik), T 138067 (Tularik), cryptophycin 52 (Eli Lilly), vinflunine (Fabre), auristatin PE (Teikoku Hormone), BMS 247550 (BMS), BMS 184476 (BMS), BMS 188797 (BMS) , taxoprexin (Protarga), SB 408075

(GlaxoSmithKline), vinorelbine, trichostatin A, E7010 (Abbott), PG-TXL (Cell Therapeutics), IDN 5109 (Bayer), A 105972 (Abbott), A 204197 (Abbott), LU 223651 (BASF), D 24851 (ASTAMedica), ER-86526 (Eisai), combretastatin A4 (BMS), isohomohalichondrin-B

(PharmaMar), ZD 6126 (AstraZeneca), AZ10992 (Asahi), IDN-5109 (Indena), AVLB (Prescient NeuroPharma), azaepothilone B (BMS), BNP-7787 (BioNumerik), CA-4 prodrug (OXiGENE), dolastatin-10 (NIH), CA-4 (OXiGENE), docetaxel, vincristine, and paclitaxel), aromatase inhibitors (e.g., aminoglutethimide, atamestane (BioMedicines), letrozole, anastrazole, YM-511 (Yamanouchi), formestane, and exemestane), thymidylate synthase inhibitors (e.g., pemetrexed (Eli Lilly), ZD-9331 (BTG), nolatrexed (Eximias), and CoFactor™ (BioKeys)), DNA antagonists (e.g., trabectedin (PharmaMar), glufosfamide (Baxter International), albumin + 32 P (Isotope Solutions), thymectacin (NewBiotics), edotreotide (Novartis), mafosfamide (Baxter International), apaziquone (Spectrum Pharmaceuticals), and 0⁶-benzylguanine (Paligent)), Farnesyltransferase inhibitors (e.g., arglabin (NuOncology Labs), lonafarnib (Schering-Plough), BAY-43-9006 (Bayer), tipifarnib (Johnson & Johnson), and perillyl alcohol (DOR BioPharma)), pump inhibitors (e.g., CBT-1 (CBA Pharma), tariquidar (Xenova), MS-209 (Schering AG), zosuquidar trihydrochloride (Eli Lilly), biricodar dicitrate (Vertex)), histone acetyltransferase inhibitors (e.g., tacedinaline (Pfizer), SAHA (Aton Pharma), MS-275 (Schering AG), pivaloyloxymethyl butyrate (Titan), depsipeptide (Fujisawa)), metalloproteinase inhibitors (e.g., Neovastat (Aeterna Laboratories), marimastat (British Biotech), CMT-3 (CollaGenex), BMS- 275291 (Celltech)), Ribonucleoside reductase inhibitors (e.g., gallium maltolate (Titan), triapine (Vion), tezacitabine (Aventis), didox (Molecules for Health)), TNFa agonists/antagonists (e.g., virulizin (Lorus Therapeutics), CDC-394 (Celgene), and revlimid (Celgene)), Endothelin A receptor antagonists (e.g., atrasentan (Abbott), ZD-4054 (AstraZeneca), and YM-598

(Yamanouchi)), Retinoic acid receptor agonists (e.g., fenretinide (Johnson & Johnson), LGD- 1550 (Ligand), and alitretinoin (Ligand)), Immuno-modulators (e.g., interferon, oncophage (Antigenics), GMK (Progenies), adenocarcinoma vaccine (Biomira), CTP-37 (AVI BioPharma), IRX-2 (Immuno-Rx), PEP-005 (Peplin Biotech), synchrovax vaccines (CTL Immuno), melanoma vaccine (CTL Immuno), p21 RAS vaccine (GemVax), dexosome therapy (Anosys), pentrix (Australian Cancer Technology), ISF-154 (Tragen), cancer vaccine (Intercell), norelin (Biostar), BLP-25 (Biomira), MGV (Progenies), β-alethine (Dovetail), and CLL therapy

(Vasogen)), hormonal and antihormonal agents (e.g., estrogens, conjugated estrogens, ethinyl estradiol, chlortrianisen, idenestrol, hydroxyprogesterone caproate, medroxyprogesterone, testosterone, testosterone propionate; fluoxymesterone, methyltestosterone, diethylstilbestrol, megestrol, bicalutamide, flutamide, nilutamide, dexamethasone , prednisone,

methylprednisolone, prednisolone, aminoglutethimide, leuprolide, octreotide, mitotane, P-04 (Novogen), 2-methoxyestradiol (EntreMed), arzoxifene (Eli Lilly), tamoxifen, toremofine, goserelin, Leuporelin, and bicalutamide), photodynamic agents (e.g., talaporfin (Light Sciences), Theralux (Theratechnologies), motexafin gadolinium (Pharaiacyclics), Pd-bacteriopheophorbide (Yeda), lutetium texaphyrin (Pharaiacyclics), and hypericin), and kinase inhibitors (e.g., imatinib (Novartis), leflunomide (Sugen/Pharmacia), ZD 1839 (AstraZeneca), erlotinib (Oncogene

Science), canertinib (Pfizer), squalamine (Genaera), SU5416 (Pharmacia), SU6668 (Pharmacia), ZD4190 (AstraZeneca), ZD6474 (AstraZeneca), vatalanib (Novartis), PKI166 (Novartis), GW2016 (GlaxoSmithKline), EKB-509 (Wyeth), trastuzumab (Genentech), OSI-774

(Tarceva™), CI-1033 (Pfizer), SU11248 (Pharmacia), RH3 (York Medical), genistein, radicinol, EKB-569 (Wyeth), kahalide F (PharmaMar), CEP-701 (Cephalon), CEP-751 (Cephalon), MLN518 (Millenium), PKC412 (Novartis), phenoxodiol (Novogen), C225 (ImClone), rhu-Mab (Genentech), MDX-H210 (Medarex), 2C4 (Genentech), MDX-447 (Medarex), ABX-EGF (Abgenix), EV1C-1C11 (ImClone), tyrphostins, gefitinib (Iressa), PTK787 (Novartis), EMD 72000 (Merck), Emodin, and Radicinol).

Other chemotherapeutic agents include SR- 27897 (CCK A inhibitor, Sanofi-Synthelabo), tocladesine (cyclic AMP agonist, Ribapharm), alvocidib (CDK inhibitor, Aventis), CV-247 (COX-2 inhibitor, Ivy Medical), P54 (COX-2 inhibitor, Phytopharm), CapCell™ (CYP450 stimulant, Bavarian Nordic), GCS-100 (gal3 antagonist, GlycoGenesys), G17DT immunogen (gastrin inhibitor, Aphton), efaproxiral (oxygenator, Alios Therapeutics), PI-88 (heparanase inhibitor, Progen), tesmilifene (histamine antagonist, YM Biosciences), histamine (histamine H2 receptor agonist, Maxim), tiazofurin (EVIPDH inhibitor, Ribapharm), cilengitide (integrin antagonist, Merck KGaA), SR-31747 (IL-1 antagonist, Sanofi-Synthelabo), CCI-779 (mTOR kinase inhibitor, Wyeth), exisulind (PDE V inhibitor, Cell Pathways), CP-461 (PDE V inhibitor, Cell Pathways), AG-2037 (GART inhibitor, Pfizer), WX-UK1 (plasminogen activator inhibitor, Wilex), PBI-1402 (PMN stimulant, ProMetic LifeSciences), bortezomib (proteasome inhibitor, Millennium), SRL-172 (T cell stimulant, SR Pharma), TLK-286 (glutathione S transferase inhibitor, Telik), PT-100 (growth factor agonist, Point Therapeutics), midostaurin (PKC inhibitor, Novartis), bryostatin-1 (PKC stimulant, GPC Biotech), CDA-II (apoptosis promotor, Everlife), SDX-101 (apoptosis promotor, Salmedix), rituximab (CD20 antibody, Genentech, carmustine, mitoxantrone, bleomycin, absinthin, chrysophanic acid, cesium oxides, ceflatonin (apoptosis promotor, ChemGenex), BCX-1777 (PNP inhibitor, BioCryst), ranpirnase

(ribonuclease stimulant, Alfacell), galarubicin (RNA synthesis inhibitor, Dong-A), tirapazamine (reducing agent, SRI International), N-acetylcysteine (reducing agent, Zambon), R-flurbiprofen (NF-kappaB inhibitor, Encore), 3CPA (NF-kappaB inhibitor, Active Biotech), seocalcitol (vitamin D receptor agonist, Leo), 131-TTM-601 (DNA antagonist, TransMolecular), eflornithine (ODC inhibitor , ILEX Oncology), minodronic acid (osteoclast inhibitor,

Yamanouchi), indisulam (p53 stimulant, Eisai), aplidine (PPT inhibitor, PharmaMar), gemtuzumab (CD33 antibody, Wyeth Ayerst), PG2 (hematopoiesis enhancer, Pharmagenesis), Immunol™ (triclosan oral rinse, Endo), triacetyluridine (uridine prodrug , Wellstat), SN-4071 (sarcoma agent, Signature Bioscience), TransMID-107™ (immunotoxin, KS Biomedix), PCK-

3145 (apoptosis promotor, Procyon), doranidazole (apoptosis promotor, Pola), CHS-828

(cytotoxic agent, Leo), trans-retinoic acid (differentiator, NIH), MX6 (apoptosis promotor, MAXIA), apomine (apoptosis promotor, ILEX Oncology), urocidin (apoptosis promotor, Bioniche), Ro-31-7453 (apoptosis promotor, La Roche), brostallicin (apoptosis promotor, Pharmacia), β-lapachone, gelonin, cafestol, kahweol, caffeic acid, and Tyrphostin AG. The invention may also use analogs of any of these agents (e.g., analogs having anticancer activity). Exemplary chemotherapeutic agents are listed in, e.g., U.S. Patent Nos. 6,864,275 and 6,984,654, hereby incorporated by reference.

Combination therapies may provide a synergistic benefit and can include sequential administration, as well as administration of these therapeutic agents in a substantially

simultaneous manner. In one example, substantially simultaneous administration is

accomplished, for example, by administering to the subject an inhibitor of NNT (e.g., an shRNA) and a second inhibitor in multiple capsules or injections at approximately the same time. The components of the combination therapies, as noted above, can be administered by the same route or by different routes (e.g., via oral administration). In different embodiments, a first inhibitor compound may be administered orally, while the one or more additional inhibitor compounds may be administered intramuscularly, subcutaneously, topically, or all therapeutic agents may be administered orally or all therapeutic agents may be administered by intravenous injection.

Subject Monitoring

The diagnostic methods described herein can also be used to monitor the progression of a disorder (e.g., a cellular proliferation disorder) during therapy or to determine the dosages of therapeutic compounds. In one embodiment, the levels of, for example, NNT polypeptides are measured repeatedly as a method of diagnosing the disorder and monitoring the treatment or management of the disorder. In order to monitor the progression of the disorder in a subject, subject samples can be obtained at several time points and may then be compared. For example, the diagnostic methods can be used to monitor subjects during chemotherapy. In this example, serum samples from a subject can be obtained before treatment with a chemotherapeutic agent, again during treatment with a chemotherapeutic agent, and again after treatment with a chemotherapeutic agent. In this example, the level of NNT in a subject is closely monitored and, if the level of NNT begins to increase during therapy, the therapeutic regimen for treatment of the disorder can be modified as determined by the clinician (e.g., the dosage of the therapy may be changed or a different therapeutic may be administered). The monitoring methods of the invention may also be used, for example, in assessing the efficacy of a particular drug or therapy in a subject, determining dosages, or in assessing progression, status, or stage of the infection.

Examples

The following Examples are intended to illustrate the invention. They are not meant to limit the invention in any way.

NNT is amplified in cancer cells

Using Tumorscape, a publicly available database designed by the Broad Institute (ΜΓΓ) to facilitate the use and understanding of high resolution copy number data amassed from multiple cancer types, we find that NNT copy number is amplified in cells presenting

proliferative disease phenotypes. NNT is amplified in nearly 1/3 (30.34%) of all cancers in the Tumorscape database (Q-value = 8.43E-6). An even greater enrichment of NNT amplification is found in the subset of Tumorscape cancers categorized as 'Lung NSC,' the non-small cell lung cancer subset. NNT amplification is found in 57.71% of NSC lung cancers (Q-value = 2.58E-6). Amongst NSC lung cancers in the Tumorscape database, NNT is focally amplified in 11.05% of cancers, and high copy number amplified (5+ copy number gain) in 8.59% of cases. NNT is located on a peak of amplification on chromosome 5p containing six genes (Fig. 1).

The amplification of NNT is accompanied by increased expression at both the mRNA and protein levels. mRNA expression is significantly elevated in NNT- amplified NSCLC cell lines in comparison to non-amplifed NSCLC cell lines (Fig. 2). NNT immunohistochemistry on normal human lung tissue demonstrates low levels of NNT in comparison to NSCLC tumor tissue, which is heavily stained (Fig. 3). Western blot analysis of NNT shows NNT protein expression in NNT-amplified and non-amplified NSCLC cell lines. The blot shows NNT to be overexpressed in particular cell lines with NNT amplification (Fig. 4). siRNA and shRNA treatments inhibit NNT in cancer cells

Identification of effective inhibitors of NNT is a critical step toward the development of an effective therapeutic. siRNA and shRNA constructs that inhibit NNT are embodiments that could contribute to the development of an effective therapeutic. We show that multiple shRNA constructs targeting NNT (shNNT) inhibit NNT expression and that siRNA constructs targeting

NNT (siNNT) inhibit NNT in multiple cancer cell types.

In an experiment showing that multiple shNNT constructs inhibit NNT expression,

H2009 cancer cells were infected with lentiviral vectors encoding either one of five tested shNNT constructs (shNNT 28489, shNNT 28507, shNNT 28512, shNNT 28513, or shNNT 28541) or a scrambled hairpin sequence (scr) control. shRNA constructs were obtained from

Open Biosystems. Lentiviruses were produced in 293T cells and cells were infected for with 293T viral supernatant + 8 μg/ml polybrene for 4 hours and selected with puromycin for 3 days. Lysates were collected following selection and examined for NNT and actin expression by western blot. Results show that each of the 5 tested shNNT constructs inhibits expression of NNT, while the scrambled hairpin sequence construct does not (Fig. 5).

In an experiment showing that siNNT inhibits NNT in multiple cancer cell types, siNNT was applied to three cancer cell types: PC9, H2009, and H1299. Cells were seeded in 12-well dishes at 100,000 cells/well. Cells were transfected with siNNT or an siRNA that did not target NNT. Transfections utilized Dharmafect 1 (Dharmacon), a transfection reagent used to attain high siRNA transfection efficiency, and 100 pmol of siRNA per treatment, with transfections carried out according to the manufacturer's instructions. Lysates were collected after 3 days. Expression of NNT was examined by western blot. Expression of actin was examined as a control. Results show that in each of these multiple cancer cell types, siNNT inhibits expression of NNT, while non-targeting siRNA constructs do not (Fig. 6). shRNA inhibition of NNT inhibits the conversion of NADH to NADPH and results in increased intracellular ROS levels.

Identification of effective inhibitors of NNT is a critical step toward the development of an effective therapeutic. Inhibition of NNT is expected to increase the ratio of NADP+ to NADPH and result in increased intracellular ROS levels. Measures of NADP+/NADPH and intracellular ROS may therefore serve as indicia of the efficacy of an NNT-inhibitor.

NNT catalyzes the conversion of NADH to NADPH, and the ratio of NADH to NADPH is therefore an indicium of NNT activity. To demonstrate that an shRNA targeting NNT could effectively inhibit NNT in cancer cells, an shRNA targeting NNT was constructed and administered to PC9 NSCLC cells, a model cancer cell line in which NNT is amplified. PC9 cells were infected with lentiviruses encoding either shRNA targeting NNT or a scrambled shRNA control. The ratio of NADP+/NADPH was determined three days after infection. The measured ratio of NADP+/NADPH was greater in the cells treated with NNT shRNA than in the control cells, demonstrating that shRNA inhibition of NNT is effective in cancer cells (Fig. 7A).

Intracellular ROS levels are a second indicium of NNT function. NADH generated by NNT can be used for biosynthetic reactions and to detoxify reactive oxygen species (ROS) generated as a byproduct of mitochondrial metabolism. To further demonstrate that an shRNA could effectively inhibit NNT, PC9 cells were infected with lentiviruses encoding either shRNA targeting NNT or a scrambled shRNA and intracellular ROS levels were determined three days after infection by DCF fluorescence. In the DCF fluorescence assay, cells are incubated with the profluorescent, lipophilic dihydrodichlorofluorescein diacetate (H2-DCF-DA), which diffuses through cell membranes and is subsequently modified in a manner that prevents its escape. Reaction of the internalized molecule with ROS results in fluorescence of DCF that

quantitatively indicates intracellular ROS levels. We show that treatment with NNT shRNA increases DCF fluorescence (i.e., increases intracellular ROS levels), further demonstrating that shRNA inhibition of NNT is effective in cancer cells (Fig. 7B).

NNT knockdown induces cell death in NNT-amplified NSCLC cell lines and inhibits proliferation of non-amplified cell lines.

The value of a cancer therapeutic is dependent upon its ability to inhibit cancer cell proliferation. A highly effective treatment may selectively kills cancer cells. To demonstrate that NNT inhibition effectively inhibits cancer cell proliferation or kills cancer cells, NSCLC cells were plated in 96- well plates, 2 x 10 cells per well, and infected on day 0 with lentiviruses encoding NNT shRNA or a scrambled shRNA control. Cell number was determined after 3, 5 and 7 days in culture by crystal violet staining and subsequent measurement of absorbance at 600 nm. Treatment with NNT shRNA in non-amplified cell lines resulted in an inhibition of growth, as the OD600 of cultures treated with NNT shRNA were lower than those of cultures treated with the scrambled shRNA control (Fig. 8). Further, treatment with NNT shRNA in NNT- amplified cell lines resulted in the death of cancer cell cultures, as demonstrated by downward growth curves (Fig. 9).

To further demonstrate the efficacy of NNT shRNA treatment of NNT-amplified cancer cells, apoptosis was assayed in PC9 cells infected with lentiviruses encoding either NNT shRNA or scrambled shRNA. Apoptosis was assayed by caspase 3/7 Glo (Promega) four days after lentiviral infection. The detection reagent fluoresces upon cleavage by casapse 3/7. Activation of caspase-3 is considered an essential event during apoptosis, and the reagent thereby provides an indicium of apoptosis. Results show a large increase in caspase 3/7 activity, demonstrating that inhibition of NNT results in the apoptotic death of cancer cells (Fig. 7C).

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims. All publications, patent applications, and patents mentioned in this specification are herein incorporated by reference to the same extent as if each independent publication, patent application, or patent was specifically and individually indicated to be incorporated by reference.

From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of this invention; can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

What is claimed is:

Claims

Claims

1. Use of a nicotinamide nucleotide transhydrogenase (NNT) gene copy number in a biological sample in a method for diagnosing a cellular proliferative disorder in a subject or assigning a prognostic risk of developing a cellular proliferative disorder in a subject, said method comprising determining a nicotinamide nucleotide transhydrogenase (NNT) gene copy number in a biological sample from said subject, wherein an amplification of the NNT gene in said biological sample from said subject relative to a control gene copy number indicates the presence of a cellular proliferative disorder in said subject or the risk of developing said cellular proliferative disorder in said subject.

2. The use of claim 1, wherein said NNT copy number is increased by at least 3-fold.

3. The use of claim 1, wherein said NNT gene copy number is determined by a hybridization- as say and/or an amplification-based assay.

4. The use of claim 1, wherein said NNT gene copy number is determined by

fluorescence in situ hybridization (FISH).

5. The use of claim 1, wherein said NNT gene copy number is determined by

comparative genomic hybridization (CGH).

6. The use of claim 1, wherein said NNT gene copy number is determined by

microarray-based CGH.

7. The use of any one of claims 1-6, wherein said cellular proliferative disorder is cancer.

8. The use of claim 7, wherein said cancer is prostate cancer, squamous cell cancer, small-cell lung cancer, non- small-cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma, gastric cancer, melanoma, or neck cancer.

9. A method for diagnosing a cellular proliferative disorder in a subject or assigning a prognostic risk of developing a cellular proliferative disorder in a subject, said method comprising determining a nicotinamide nucleotide transhydrogenase (NNT) gene copy number in a biological sample from said subject, wherein an amplification of the NNT gene in said biological sample from said subject relative to a control gene copy number indicates the presence of a cellular proliferative disorder in said subject or the risk of developing said cellular proliferative disorder in said subject.

10. The method of claim 9, wherein said NNT copy number is increased by at least 3- fold.

11. The method of claim 9, wherein said NNT gene copy number is determined by a hybridization- as say and/or an amplification-based assay.

12. The method of claim 9, wherein said NNT gene copy number is determined by fluorescence in situ hybridization (FISH).

13. The method of claim 9, wherein said NNT gene copy number is determined by comparative genomic hybridization (CGH).

14. The method of claim 9, wherein said NNT gene copy number is determined by microarray-based CGH.

15. A method of identifying an inhibitor of nicotinamide nucleotide transhydrogenase (NNT), said method comprising:

(a) providing a cancer cell that expresses NNT or a functional fragment thereof.

(b) contacting a said cell with a candidate compound; and

(c) determining a level of NADPH present in said cell contacted with said candidate compound, wherein a reduction in the level of NADPH in said cell contacted with said candidate compound compared to a level of NADPH in a control cell not contacted with said candidate compound identifies said candidate compound as an inhibitor of NNT.

16. A method of identifying an inhibitor of nicotinamide nucleotide transhydrogenase (NNT), said method comprising: (a) providing a cancer cell comprising NNT, or a functional fragment thereof, and

NADP⁺.

(b) contacting said cancer cell with a candidate compound; and

(c) determining a level of NADPH present in said cancer cell, wherein a reduction in the level of NADPH in said sample contacted with said candidate compound compared to a level of NADPH in a control sample not contacted with said candidate compound identifies said candidate compound as an inhibitor of NNT.

17. The method of any one of claims 15-16, wherein said determining step is performed using fluorescence spectroscopy.

18. A method of treating or reducing the likelihood of developing a cellular proliferative disorder in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of an inhibitor of nicotinamide nucleotide transhydrogenase (NNT).

19. A method of treating or reducing the likelihood of developing a cellular proliferative disorder in a subject in need thereof characterized by an amplification of nicotinamide nucleotide transhydrogenase (NNT) gene, said method comprising administering to said subject a therapeutically effective amount of an inhibitor of NNT.

20. The method of claim 18 or 19, wherein said inhibitor of NNT reduces or inhibits the activity or expression levels of an NNT polypeptide or nucleic acid molecule.

21. The method of claim 20, wherein said activity of said NNT polypeptide is the conversion of NADP⁺ to NADPH or the promotion of cell proliferation.

22. The method of claim 18 or 19, wherein said inhibitor of NNT is a peptide, nucleic acid molecule, aptamer, small molecule, or polysaccharide.

23. The method of claim 22, wherein said nucleic acid molecule is short interfering RNA (siRNA) or microRNA.

24. The method of claim 18 or 19, wherein said inhibitor of NNT is FSB A, NN'- dicyclohexylcarbodi-imide or butane-2,3-dione.

25. The method of claim 18 or 19, further comprising administering to said subject an additional therapeutic agent.

26. The method of claim 25, wherein said additional therapeutic agent is a

chemotherapeutic agent.

27. The method of any one of claims 9-26, wherein said cellular proliferative disorder is cancer.

28. The method of claim 27, wherein said cancer is prostate cancer, squamous cell cancer, small-cell lung cancer, non- small-cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,

gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, vulval cancer, thyroid cancer, hepatic carcinoma, gastric cancer, melanoma, or neck cancer.

29. Use of an inhibitor of nicotinamide nucleotide transhydrogenase (NNT) for treating or reducing the likelihood of developing a cellular proliferative disorder in a subject in need thereof, said use comprising administering to said subject a therapeutically effective amount of an inhibitor of NNT.

30. Use of an inhibitor of nicotinamide nucleotide transhydrogenase (NNT) for treating or reducing the likelihood of developing a cellular proliferative disorder characterized by an amplification of an NNT gene, said use comprising administering to a subject in need thereof a therapeutically effective amount of an inhibitor of NNT.