METHODS AND KITS USED IN IDENTIFYING GLIOBLASTOMA

INVENTOR NHAN TRAN

BACKGROUND OF THE INVENTION

Glioblastoma multiforme (GBM) (also referred to interchangeably as glioma) is the most malignant form of all primary adult brain tumors (See Reference 1) Although significant technical advances in surgical and radiation treatment for brain tumors have emerged, their impact on clinical outcome for patients has been only modest (See References 2-4). Of the features that characterize GBM, arguably none is more clinically significant than the propensity of glioma cells to infiltrate into normal brain tissue. These invasive cells render complete resection impossible and confer resistance to chemo- and radiation-therapy. Thus, improved treatment of malignant glioma awaits a way of targeting the dispersing tumor cells in the CNS.

BRIEF SUMMARY OF THE INVENTION

It is an object of the invention to identify a tumor as an invasive glioblastoma.

It is an object of the invention to predict the survival time of a patient with glioblastoma. It is an object of the invention to treat patients with glioblastoma.

It is an object of the invention to provide kits that facilitate the identification of invasive glioblastoma.

It is an object of the invention to provide a personalized medicine based test used in staging glioblastoma patients for treatment.

The above and other objects may be achieved through the use of methods involving, obtaining a sample of a tumor from a subject, adding a first reagent capable of binding to a marker selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2 to a mixture comprising the sample; subjecting the mixture to conditions that allow detection of the binding of the reagent to the marker; and classifying the tumor into a cohort selected from the group consisting of invasive glioblastoma and proliferative glioblastoma on the basis of a result of the binding of the reagent to the sample. In some aspects of the invention, the marker comprises SEQ ID NO. 2. In those aspects, the first reagent may comprise a first antibody. The first antibody may comprise a first label. The first label may be any label, such as a fluorescent compound, an enzyme, a radioisotope, or a ligand. The method may further comprise adding a second antibody capable of binding to the first antibody to the mixture. The second antibody may comprise a second label. The second label may be any label such as a fluorescent compound, an enzyme, a radioisotope, or a ligand.

In other aspects of the invention, the marker may comprise SEQ ID NO. 1. In those other aspects, the first reagent may comprise a first nucleic acid and the first nucleic acid may further comprise a first oligonucleotide capable of binding to part of SEQ ID NO. 1. The method may further comprise purifying RNA from the sample, performing reverse transcription on the RNA, adding a second oligonucleotide capable of binding to part of SEQ ID NO. 1 to the mixture, wherein the conditions comprise subjecting the mixture to nucleic acid amplification, wherein the first oligonucleotide and the second oligonucleotide are capable of binding to different sequences on SEQ ID NO. 1, and wherein the first oligonucleotide and the second

oligonucleotide are capable of binding to separate nucleic acid strands. The first oligonucleotide may be any oligonucleotide such as an oligonucleotide that includes SEQ ID NO. 3. The second oligonucleotide may be any oligonucleotide including an oligonucleotide that includes SEQ ID NO. 4. The method may further comprise adding a third oligonucleotide to the mixture, wherein the third oligonucleotide is capable of binding to part of SEQ ID NO. 1 between the sequences to which the first oligonucleotide and the second oligonucleotide are capable of binding. The third oligonucleotide may comprise a fluorescent compound. The fluorescent compound may be any fluorescent compound including a compound selected from the group consisting of FAM, dRl 10, 5 -FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, BHQ+, Gold540, and LIZ. The method may comprise performing DNA sequencing on a product of the nucleic acid amplification. In some aspects of the invention, the first reagent may be affixed to a solid substrate. In those aspects, the conditions may comprise microarray analysis. The sample may be any sample such as a sample that comprises a cell. One example of such a sample is a brain biopsy sample.

The above and other objects may be achieved through the use of methods involving obtaining a sample of a tumor from a patient, adding a first reagent capable of binding to a marker selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2 to a mixture comprising the sample, subjecting the mixture to conditions that allow detection of the binding of the reagent to the marker, and classifying the patient into a cohort selected from the group consisting of short term survivors and long term survivors. The short-term survivors may be predicted to survive less than 680 days from biopsy including less than 400 days from biopsy. The long-term survivors may be predicted to survive more than 680 days from biopsy, including more than 950 days from biopsy.

The above and other objects may be achieved through the use of methods involving: obtaining a sample of a tumor from a patient, adding a first reagent capable of binding to a marker selected from a group consisting of SEQ ID NO. 1 and SEQ ID NO. 2 to a mixture comprising the sample, subjecting the mixture to conditions that allow detection of the binding of the reagent to the marker, and treating the patient on the basis of a result of the binding of the reagent to the sample. The result may be the expression of the marker below a threshold level and treating the patient may comprise administering a therapeutic composition comprising a compound selected from the group consisting of temozolimide and bevacizumab. The result may be expression of the marker above a threshold level and treating the patient may comprise administering a therapeutic composition from a class selected from the group consisting of TROY inhibitors, Pyk2 inhibitors, Racl inhibitors, Dockl80 inhibitors, Dock7 inhibitors, TWEAK inhibitors, Fnl4 inhibitors, BAD inhibitors, and PI3K inhibitors.

The above and other objects may be achieved through the use of kits involving a first reagent capable of specific binding to a marker selected from the group consisting of SEQ ID NO. 1 and SEQ ID NO. 2 and an indication of a result, wherein the result signifies that the tumor is an invasive glioblastoma. The first reagent may comprise a first antibody. The first antibody may comprise a first label. The first label may be any label such as a fluorescent compound, an enzyme, a radioisotope, or a ligand. The kit may further comprise a second antibody capable of binding to the first antibody. The second antibody may comprise a second label. The second label may be any label such as a fluorescent compound, an enzyme, a radioisotope, or a ligand. The first reagent may comprise a first nucleic acid. The first nucleic acid may comprise a first oligonucleotide capable of binding to part of SEQ ID NO. 1. The first oligonucleotide may be any oligonucleotide meeting this description, including SEQ ID NO. 3 and SEQ ID NO. 4. The kit may further comprise a second oligonucleotide capable of binding to part of SEQ ID NO. 1 wherein the first oligonucleotide and the second oligonucleotide are capable of binding to different sequences on SEQ ID NO. 1 and wherein the first oligonucleotide and the second oligonucleotide are capable of binding to separate nucleic acid strands. The kit may further comprise a third nucleotide wherein the third nucleotide binds to part of SEQ ID NO. 1 between the sequences to which the first oligonucleotide and the second oligonucleotide are capable of binding. The third nucleic acid may comprise a fluorescent compound. The florescent compound may be any fluorescent compound including dRl 10, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, BHQ+, Gold540, and LIZ. The kit may further comprise an enzyme. The enzyme may be any enzyme such as a DNA polymerase or a reverse transcriptase. The first reagent may be affixed to a solid substrate. The indication may be any indication, such as a positive control or a writing. A writing may be made available via a website, or it may include a photograph. The indication may be physically included in the kit. The indication may comprise software configured to detect a level of expression as input and identification of invasive glioblastoma as output. The software may be incorporated into a machine configured to detect binding of the reagent to the marker.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts expression of TROY in normal brain and various glioblastoma types in the NCBI Gene Expression Omnibus GDS1962 dataset.

Figure 2 depicts expression of TROY in a separate set of normal brain and various glioblastoma types by QRT-PCR.

Figure 3 depicts TROY expression data from the NCBI Gene Expression Omnibus

GDS1962 dataset with tissues grouped as long-term and short-term survivors.

Figure 4 depicts a Western blot showing expression of TROY in four glioblastoma cell lines.

Figure 5 depicts a Western blot showing suppression of TROY expression in two of the cell types using siRNA that targets TROY expression.

Figure 6 depicts a graph showing significantly slowed migration of four glioblastoma cell lines when those lines are transfected with siRNA targeting TROY.

Figure 7 depicts expression of a construct comprising HA-tagged TROY transfected into two glioblastoma cell lines.

Figure 8 depicts increased migration rate of cell lines transfected with the HA-tagged

TROY construct. Figure 9 depicts increased depth of invasion into rat brain slices by a GFP-tagged TROY transfected cell line.

Figure 10 depicts immuno fluorescent staining for HA in HA-tagged TROY transfected cell lines.

Figure 11 depicts (top) a Western Blot showing cell lysates from a glioblastoma cell line transfected with Pyk2 or HA-tagged TROY as indicated and (bottom) a Western blot of immunoprecipitates with anti-HA antibodies that show an association of Pyk2 with TROY.

Figure 12 depicts increased migration rate in a glioblastoma cell line transfected with HA-tagged TROY that is slowed when Pyk2 expression is suppressed.

Figure 13 depicts reduced migration rate in a glioblastoma cell line transfected with a dominant-negative Pyk2 construct, whether or not the TROY expression is endogenous or from HA-tagged TROY.

Figure 14 depicts a Western blot showing increased phosphorylation of RhoA when TROY expression is suppressed and reduced phosphorylation of Rac-1 when TROY expression is suppressed.

Figure 15 depicts a Western blot showing increased phosphorylation of Rac-1 when a cell line is transfected with HA-tagged TROY. This effect is reduced when Pyk2 expression is suppressed.

Figure 16 depicts reduced migration of a glioblastoma cell line when Rac-1 expression is suppressed - whether or not the cell line expresses endogenous or HA-tagged TROY.

Figure 17 depicts a Western blot validation of suppression of Racl expression by Racl siRNA.

Figure 18 depicts increased Akt, IkBa, and Erkl/2 phosphorylation when a glioblastoma cell line is transfected with HA-tagged TROY.

Figure 19 depicts increased sensitivity of a glioblastoma cell line to temozolimide when TROY expression is suppressed.

Figure 20 depicts reduced sensitivity of a glioblastoma cell line to temozolimide when the cell line is transfected with a HA-tagged TROY construct.

Figure 21 depicts reduced migration of a glioblastoma cell line when Dockl80 and Dock7 expression are suppressed. Figure 22 depicts reduced depth of invasion of glioblastoma cells when Dock7 expression is suppressed using two different siRNAs.

Figure 23 depicts a Western blot showing TROY expression in glioblastoma xenografts grown in murine brain.

DETAILED DESCRIPTION OF THE INVENTION

Gliomas also called glioblastomas, primary brain tumors that derive from glial support cells, are the most common primary tumor of the adult central nervous system and will result in an estimated 13,000 deaths in 2010 (See References 1, 3, 11, and 12). Adult gliomas of astrocytic origin (astrocytomas) comprise a spectrum of neoplasms that are generally classified by WHO standards into low-grade benign tumors (i.e. juvenile pilocytic astrocytoma, diffuse astrocytoma) and high-grade malignant tumors (i.e. anaplastic astrocytoma and glioblastoma multiforme; GBM). Patients diagnosed with grade IV GBM, the most aggressive malignant glioma, have a median survival of 9-12 months after the onset of clinical symptoms (See References 11-13). Molecular analyses of glioma specimens have identified several common genetic alterations (e.g., pl6INK4a deletion) and gene expression changes (e.g., EGFR overexpression) that may contribute to glioblastoma formation (See References 14 and 15).

In general, gliomas are extremely difficult to treat using conventional approaches (See References 12-16.) This is primarily due to the intrinsic propensity of glioma cells to exit the tumor core and invade the adjacent normal brain parenchyma (See References 3 and 4). These migrating cells escape surgical resection and are poorly targeted by radiation or chemotherapy. They sometimes travel over long distances, frequently along blood vessel and fiber tracts, and then initiate secondary tumor growth at their final destination. This distinguishing invasive ability is not shared by nonglial cells that metastasize from other primary tumor sites (e.g. breast) to brain tissue. The invasion of glioma cells is likely triggered by a presently undefined signal or signals that promote a cascade of cellular responses, including cell elongation, integrin-mediated cell attachment to extracellular matrix (ECM) molecules, the production and secretion of ECM- degrading enzymes, and cell movement (See References 17 and 18).

Migrating glioma cells exhibit decreased susceptibility to pro-apoptotic agents (See Reference 19) providing them with an additional mechanism for resisting current radiological and chemotherapeutic treatment modalities. TROY (TNFRSF19) is an orphan member of the TNFR superfamily that is highly expressed in embryonic and adult CNS, and developing hair follicles (See References 5-10). During mouse embryogenesis, TROY mRNA is detected in many developing tissues including the limb buds, eyelids, whiskers, mammary glands, epidermis, bronchial, tongue, dental and gastric epithelium as well as the germinal zones of the CNS including the ventricular zone and subventricular zone. However, in adult animals, TROY expression changes and is primarily restricted to hair follicles and neuron-like cells in the cerebrum, cerebral cortex, developing olfactory system as well as dorsal root and retinal ganglion neurons (See References 5-10) In the peripheral nervous system, TROY functions as a co-receptor for the ligand-binding Nogo-66 receptor 1 (NgRl) to form the TROY/NgRl /LINGO complex that activates the RhoA pathway to inhibit neurite outgrowth of dorsal root ganglion neurons in adult mice (See References 6 and 9). In humans, TROY mRNA is primarily expressed in the brain and also the prostate, whereas low or undetectable levels are observed in the heart, lung, liver, thymus, uterus, skeletal muscle, spleen, colon testis, kidney and peripheral blood lymphocytes (See Reference 20). The reason or mechanism for this "switch-off of TROY expression after birth is unclear, but its strict control indicates that aberrant expression may be detrimental. Indeed, it has been recently reported that TROY is highly expressed in primary and metastatic melanoma cells, but not in melanocytes found in normal skin biopsies and primary skin cell cultures (See Reference 21).

Herein, the Inventor demonstrates that TROY serves as a target or marker of invasive glioblastoma, that its expression is linked to poor therapeutic outcome and that it serves as a marker of resistance to temozolimide and as a marker of sensitivity to classes of drugs that treat glioblastoma by targeting pathways that contribute to glioma cell migration and invasion.

A marker may be any molecular structure produced by a cell, expressed inside the cell, accessible on the cell surface, or secreted by the cell. A marker may be any protein,

carbohydrate, fat, nucleic acid, catalytic site, or any combination of these such as an enzyme, glycoprotein, cell membrane, virus, cell, organ, organelle, or any uni- or multimolecular structure or any other such structure now known or yet to be disclosed whether alone or in combination. A marker may also be called a target and the terms are used interchangeably.

A marker may be represented by the sequence of a nucleic acid from which it can be derived. Examples of such nucleic acids include miRNA, tRNA, siRNA, mRNA, cDNA, or genomic DNA sequences. While a marker may be represented by the sequence of a single nucleic acid strand (e.g. 5' 3'), nucleic acid reagents that bind the marker may also bind to the complementary strand (e.g. 3' 5'). Alternatively, a marker may be represented by a protein sequence. The concept of a marker is not limited to the products of the exact nucleic acid sequence or protein sequence by which it may be represented. Rather, a marker encompasses all molecules that may be detected by a method of assessing the expression of the marker.

Examples of molecules encompassed by a marker include point mutations, silent mutations, deletions, frameshift mutations, translocations, alternative splicing derivatives, differentially methylated sequences, differentially modified protein sequences, truncations, soluble forms of cell membrane associated markers, and any other variation that results in a product that may be identified as the marker. The following nonlimiting examples are included for the purposes of clarifying this concept: If expression of a specific marker in a sample is assessed by RTPCR, and if the sample expresses an mRNA sequence different from the sequence used to identify the specific marker by one or more nucleotides, but the marker may still be detected using RTPCR, then the specific marker encompasses the sequence present in the sample. Alternatively if expression of a specific marker in a sample is assessed by an antibody and the amino acid sequence of the marker in the sample differs from a sequence used to identify marker by one or more amino acids, but the antibody is still able to bind to the version of the marker in the sample, then the specific marker encompasses the sequence present in the sample.

Expression encompasses any and all processes through which material derived from a nucleic acid template may be produced. Expression thus includes processes such as RNA transcription, mRNA splicing, protein translation, protein folding, post-translational

modification, membrane transport, associations with other molecules, addition of carbohydrate moeties to proteins, phosphorylation, protein complex formation and any other process along a continuum that results in biological material derived from genetic material whether in vitro, in vivo, or ex vivo. Expression also encompasses all processes through which the production of material derived from a nucleic acid template may be actively or passively suppressed. Such processes include all aspects of transcriptional and translational regulation. Examples include heterochromatic silencing, differential methylation, transcription factor inhibition, any form of RNAi silencing, microRNA silencing, alternative splicing, protease digestion, posttranslational modification, and alternative protein folding.

Expression may be assessed by any number of methods used to detect material derived from a nucleic acid template used currently in the art and yet to be developed. Examples of such methods include any nucleic acid detection method including the following nonlimiting examples, microarray analysis, RNA in situ hybridization, R Ase protection assay, Northern blot, reverse transcriptase PCR, quantitative PCR, quantitative reverse transcriptase PCR, quantitative real-time reverse transcriptase PCR, reverse transcriptase treatment followed by direct sequencing, direct sequencing of genomic DNA, or any other method of detecting a specific nucleic acid now known or yet to be disclosed. Other examples include any process of assessing protein expression including flow cytometry, immunohistochemistry, ELISA, Western blot, and immunoaffinity chromatograpy, HPLC, mass spectrometry, protein microarray analysis, PAGE analysis, isoelectric focusing, 2-D gel electrophoresis, or any enzymatic assay. Methods of detecting expression may include methods of purifying nucleic acid, protein, or some other material depending on the type of marker. Any method of nucleic acid purification may be used, depending on the type of marker. Examples include phenol alcohol extraction, ethanol extraction, guanidium isothionate extraction, gel purification, size exclusion chromatography, cesium chloride preparations, and silica resin preparation. Any method of protein purification may be used, also depending on the type of marker. Examples include size exclusion

chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatograpy (including affinity chromatography of tagged proteins), metal binding, immunoaffinity chromatography, and HPLC.

Nucleic acid amplification is a process by which copies of a nucleic acid may be made from a source nucleic acid. Nucleic acids that may be subjected to amplification may be from any source. In some nucleic amplification methods, the copies are generated exponentially. Examples of nucleic acid amplification include but are not limited to: the polymerase chain reaction (PCR), ligase chain reaction (LCR,) self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA,) strand displacement amplification (SDA,) amplification with Q replicase, whole genome amplification with enzymes such as φ29, whole genome PCR, in vitro transcription with any RNA polymerase, or any other method by which copies of a desired sequence are generated.

Polymerase chain reaction (PCR) is a particular method of amplifying DNA, generally involving the mixing of a nucleic sample, two or more primers, a DNA polymerase, which may be a thermostable DNA polymerase such as Taq or Pfu, and deoxyribose nucleoside triphosphates (dNTP's). In general, the reaction mixture is subjected to temperature cycles comprising a denaturation stage, (typically 80-100 C) an annealing stage with a temperature that is selected based on the melting temperature (Tm) of the primers and the degeneracy of the primers, and an extension stage (for example 40-75 C.) In real-time PCR analysis, additional reagents, methods, optical detection systems, and devices are used that allow a measurement of the magnitude of fluorescence in proportion to concentration of amplified DNA. In such analyses, incorporation of fluorescent dye into the amplified strands may be detected or labeled probes that bind to a specific sequence during the annealing phase release their fluorescent tags during the extension phase. Either of these will allow a quantification of the amount of specific DNA present in the initial sample. Often, the result of a real-time PCR will be expressed in the terms of cycle threshold (Ct) values. The Ct represents the number of PCR cycles for the fluorescent signal from a real-time PCR reaction to cross a threshold value of fluorescence. Ct is inversely proportional to the amount of target nucleic acid originally present in the sample. RNA may be detected by PCR analysis by creating a DNA template from RNA through a reverse transcriptase enzyme.

Other methods used to assess expression include the use of natural or artificial ligands capable of specifically binding a marker. Such ligands include antibodies, antibody complexes, conjugates, natural ligands, small molecules, nanoparticles, or any other molecular entity capable of specific binding to a marker. Antibodies may be monoclonal, polyclonal, or any antibody fragment including a Fab, F(ab)2, Fv, scFv, phage display antibody, peptibody, multispecific ligand, or any other reagent with specific binding to a marker. Ligands may be associated with a label such as a radioactive isotope or chelate thereof, dye (fluorescent or nonfiuorescent,) stain, enzyme, metal, or any other substance capable of aiding a machine or a human eye from differentiating a cell expressing a marker from a cell not expressing a marker. Additionally, expression may be assessed by monomeric or multimeric ligands associated with substances capable of killing the cell. Such substances include protein or small molecule toxins, cytokines, pro-apoptotic substances, pore forming substances, radioactive isotopes, or any other substance capable of killing a cell.

Differential expression encompasses any detectable difference between the expression of a marker in one sample relative to the expression of the marker in another sample. Differential expression may be assessed by a detector, an instrument containing a detector, or by aided or unaided human eye. Examples include but are not limited to differential staining of cells in an IHC assay configured to detect a marker, differential detection of bound RNA on a microarray to which a sequence capable of binding to the marker is bound, differential results in measuring RTPCR measured in the number of PCR cycles necessary to reach a particular optical density at a wavelength at which a double stranded DNA binding dye (e.g. SYBR Green) incorporates, differential results in measuring label from a reporter probe used in a real-time RTPCR reaction, differential detection of fluorescence on cells using a flow cytometer, differential intensities of bands in a Northern blot, differential intensities of bands in an RNAse protection assay, differential cell death measured by apoptotic markers, differential cell death measured by shrinkage of a tumor, or any method that allows a detection of a difference in signal between one sample or set of samples and another sample or set of samples.

The expression of the marker in a sample may be compared to a level of expression predetermined to predict the presence or absence of a particular physiological characteristic. The level of expression may be derived from a single control or a set of controls. A control may be any sample with a previously determined level of expression. A control may comprise material within the sample or material from sources other than the sample. Alternatively, the expression of a marker in a sample may be compared to a control that has a level of expression

predetermined to signal or not signal a cellular or physiological characteristic. This level of expression may be derived from a single source of material including the sample itself or from a set of sources. Comparison of the expression of the marker in the sample to a particular level of expression results in a prediction that the sample exhibits or does not exhibit the cellular or physiological characteristic.

Prediction of a cellular or physiological characteristic includes the prediction of any cellular or physiological state that may be predicted by assessing the expression of a marker. Examples include the identity of a cell as a particular cell including a particular normal or cancer cell type, the likelihood that one or more diseases is present or absent, the likelihood that a present disease will progress, remain unchanged, or regress, the likelihood that a disease will respond or not respond to a particular therapy, or any other outcome. Further examples include the likelihood that a cell will move, senesce, apoptose, differentiate, metastasize, or change from any state to any other state or maintain its current state.

Expression of a marker in a sample may be more or less than that of a level predetermined to predict the presence or absence of a cellular or physiological characteristic. The expression of the marker in the sample may be more than Ι,ΟΟΟ,ΟΟΟχ, ΙΟΟ,ΟΟΟχ, ΙΟ,ΟΟΟχ, ΙΟΟΟχ, ΙΟΟχ, lOx, 5x, 2x, lx, 0.5x, O. lx O.Olx, O.OOlx, O.OOOlx, O.OOOOlx, O.OOOOOlx, O.OOOOOOlx or less than that of a level predetermined to predict the presence or absence of a cellular or physiological characteristic.

The invention contemplates assessing the expression of the marker in any biological sample from which the expression may be assessed. One skilled in the art would know to select a particular biological sample and how to collect said sample depending upon the marker that is being assessed. Examples of sources of samples include but are not limited to biopsy or other in vivo or ex vivo analysis of prostate, breast, skin, muscle, fascia, brain, endometrium, lung, head and neck, pancreas, small intestine, blood, liver, testes, ovaries, colon, skin, stomach, esophagus, spleen, lymph node, bone marrow, kidney, placenta, or fetus. In some aspects of the invention, the sample comprises a fluid sample, such as peripheral blood, lymph fluid, ascites, serous fluid, pleural effusion, sputum, cerebrospinal fluid, amniotic fluid, lacrimal fluid, stool, or urine.

Samples include single cells, whole organs or any fraction of a whole organ, in any condition including in vitro, ex vivo, in vivo, post-mortem, fresh, fixed, or frozen.

One type of cellular or physiological characteristic is the risk that a particular disease outcome will occur. Assessing this risk includes the performing of any type of test, assay, examination, result, readout, or interpretation that correlates with an increased or decreased probability that an individual has had, currently has, or will develop a particular disease, disorder, symptom, syndrome, or any condition related to health or bodily state. Examples of disease outcomes include, but need not be limited to survival, death, progression of existing disease, remission of existing disease, initiation of onset of a disease in an otherwise disease-free subject, or the continued lack of disease in a subject in which there has been a remission of disease. Assessing the risk of a particular disease encompasses diagnosis in which the type of disease afflicting a subject is determined. Assessing the risk of a disease outcome also encompasses the concept of prognosis. A prognosis may be any assessment of the risk of disease outcome in an individual in which a particular disease has been diagnosed. Assessing the risk further encompasses prediction of therapeutic response in which a treatment regimen is chosen based on the assessment. Assessing the risk also encompasses a prediction of overall survival after diagnosis. Determining the level of expression that signifies a physiological or cellular characteristic may be assessed by any of a number of methods. The skilled artisan will understand that numerous methods may be used to select a level of expression for a particular marker or a plurality of markers that signifies a particular physiological or cellular characteristics. In diagnosing the presence of a disease, a threshold value may be obtained by performing the assay method on samples obtained from a population of patients having a certain type of disease (cancer for example,) and from a second population of subjects that do not have the disease. In assessing disease outcome or the effect of treatment, a population of patients, all of which have, a disease such as cancer, may be followed for a period of time. After the period of time expires, the population may be divided into two or more groups. For example, the population may be divided into a first group of patients whose disease progresses to a particular endpoint and a second group of patients whose disease does not progress to the particular endpoint. Examples of endpoints include disease recurrence, death, metastasis or other states to which disease may progress. If expression of the marker in a sample is more similar to the predetermined expression of the marker in one group relative to the other group, the sample may be assigned a risk of having the same outcome as the patient group to which it is more similar.

In addition, one or more levels of expression of the marker may be selected that signify a particular physiological or cellular characteristic. For example, Receiver Operating

Characteristic curves, or "ROC" curves, may be calculated by plotting the value of a variable versus its relative frequency in two populations. For any particular marker, a distribution of marker expression levels for subjects with and without a disease may overlap. This indicates that the test does not absolutely distinguish between the two populations with complete accuracy. The area of overlap indicates where the test cannot distinguish the two groups. A threshold is selected. Expression of the marker in the sample above the threshold indicates the sample is similar to one group and expression of the marker below the threshold indicates the sample is similar to the other group. The area under the ROC curve is a measure of the probability that the expression correctly indicated the similarity of the sample to the proper group. See, e.g., Hanley et ah, Radiology 143: 29-36 (1982) hereby incorporated by reference.

Additionally, levels of expression may be established by assessing the expression of a marker in a sample from one patient, assessing the expression of additional samples from the same patient obtained later in time, and comparing the expression of the marker from the later samples with the initial sample or samples. This method may be used in the case of markers that indicate, for example, progression or worsening of disease or lack of efficacy of a treatment regimen or remission of a disease or efficacy of a treatment regimen.

Other methods may be used to assess how accurately the expression of a marker signifies a particular physiological or cellular characteristic. Such methods include a positive likelihood ratio, negative likelihood ratio, odds ratio, and/or hazard ratio. In the case of a likelihood ratio, the likelihood that the expression of the marker would be found in a sample with a particular cellular or physiological characteristic is compared with the likelihood that the expression of the marker would be found in a sample lacking the particular cellular or physiological characteristic.

An odds ratio measures effect size and describes the amount of association or non- independence between two groups. An odds ratio is the ratio of the odds of a marker being expressed in one set of samples versus the odds of the marker being expressed in the other set of samples. An odds ratio of 1 indicates that the event or condition is equally likely to occur in both groups. An odds ratio grater or less than 1 indicates that expression of the marker is more likely to occur in one group or the other depending on how the odds ratio calculation was set up.

A hazard ratio may be calculated by estimate of relative risk. Relative risk is the chance that a particular event will take place. It is a ratio of the probability that an event such as development or progression of a disease will occur in samples that exceed a threshold level of expression of a marker over the probability that the event will occur in samples that do not exceed a threshold level of expression of a marker. Alternatively, a hazard ratio may be calculated by the limit of the number of events per unit time divided by the number at risk as the time interval decreases. In the case of a hazard ratio, a value of 1 indicates that the relative risk is equal in both the first and second groups. A value greater or less than 1 indicates that the risk is greater in one group or another, depending on the inputs into the calculation.

Additionally, multiple threshold levels of expression may be determined. This can be the case in so-called "tertile," "quartile," or "quintile" analyses. In these methods, multiple groups can be considered together as a single population, and are divided into 3 or more bins having equal numbers of individuals. The boundary between two of these "bins" may be considered threshold levels of expression indicating a particular level of risk of a disease developing or signifying a physiological or cellular state. A risk may be assigned based on which "bin" a test subject falls into. A subject includes any human or non-human mammal, including for example: a primate, cow, horse, pig, sheep, goat, dog, cat, or rodent, capable of developing cancer including human patients that are suspected of having cancer, that have been diagnosed with cancer, or that have a family history of cancer. Methods of identifying subjects suspected of having cancer include but are not limited to: physical examination, family medical history, subject medical history including exposure to environmental factors, biopsy, or any of a number of imaging technologies such as ultrasonography, computed tomography, magnetic resonance imaging, magnetic resonance spectroscopy, or positron emission tomography.

Cancer cells include any cells derived from a tumor, neoplasm, cancer, precancer, cell line, malignancy, or any other source of cells that have the potential to expand and grow to an unlimited degree. Cancer cells may be derived from naturally occurring sources or may be artificially created. Cancer cells may also be capable of invasion into other tissues and metastasis. Cancer cells further encompass any malignant cells that have invaded other tissues and/or metastasized. One or more cancer cells in the context of an organism may also be called a cancer, tumor, neoplasm, growth, malignancy, or any other term used in the art to describe cells in a cancerous state.

Examples of cancers that could serve as sources of cancer cells include solid tumors such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, throat 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, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung carcinoma, bladder carcinoma, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, and retinoblastoma. Additional cancers that may serve as sources of cancer cells include blood borne cancers such as acute lymphoblastic leukemia ("ALL,"), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia ("AML"), acute promyelocytic leukemia ("APL"), acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia ("CML"), chronic lymphocytic leukemia ("CLL"), hairy cell leukemia, multiple myeloma, lymphoblastic leukemia,

myelogenous leukemia, lymphocytic leukemia, myelocytic leukemia, Hodgkin's disease, non- Hodgkin's Lymphoma, Waldenstrom's macroglobulinemia, Heavy chain disease, and

Polycythemia vera.

The present invention further provides kits to be used in assessing the expression of a particular R A in a sample from a subject to assess the risk of developing disease. Kits include any combination of components that facilitates the performance of an assay. A kit that facilitates assessing the expression of an RNA may include suitable nucleic acid-based and immunological reagents as well as suitable buffers, control reagents, and printed protocols.

Kits that facilitate nucleic acid based methods may further include one or more of the following: specific nucleic acids such as oligonucleotides, labeling reagents, enzymes including PCR amplification reagents such as Taq or Pfu; reverse transcriptase, or one or more other polymerases, and/or reagents that facilitate hybridization. Specific nucleic acids may include nucleic acids, polynucleotides, oligonucleotides (DNA, or RNA), or any combination of molecules that includes one or more of the above, or any other molecular entity capable of specific binding to a nucleic acid marker. In one aspect of the invention, the specific nucleic acid comprises one or more oligonucleotides capable of hybridizing to the marker.

A specific nucleic acid may include a label. A label may be any substance capable of aiding a machine, detector, sensor, device, or enhanced or unenhanced human eye from differentiating a sample that that displays positive expression from a sample that displays reduced expression. Examples of labels include but are not limited to: a radioactive isotope or chelate thereof, a dye (fluorescent or nonfiuorescent,) stain, enzyme, or nonradioactive metal. Specific examples include but are not limited to: fluorescein, biotin, digoxigenin, alkaline phosphatase, biotin, streptavidin, 3 H, 14 C, 32 P, 35 S, or any other compound capable of emitting radiation, rhodamine, 4-(4'-dimethylaminophenylazo) benzoic acid ("Dabcyl"); 4-(4'- dimethylamino-phenylazo)sulfonic acid (sulfonyl chloride) ("Dabsyl"); 5-((2-aminoethyl)- amino)-naphtalene-l -sulfonic acid ("EDANS"); Psoralene derivatives, haptens, cyanines, acridines, fluorescent rhodol derivatives, cholesterol derivatives; ethylenediaminetetraaceticacid ("EDTA") and derivatives thereof or any other compound that signals the presence of the labeled nucleic acid. In one embodiment of the invention, the label includes one or more dyes optimized for use in genotyping. Examples of such dyes include but are not limited to: dRUO, 5-FAM, 6FAM, dR6G, JOE, HEX, VIC, TET, dTAMRA, TAMRA, NED, dROX, PET, and LIZ.

An oligonucleotide is a reagent capable of binding a nucleic acid sequence. An oligonucleotide may be any polynucleotide of at least 2 nucleotides. Oligonucleotides may be less than 10, less than 15, less than 20, less than 30, less than 40, less than 50, less than 75, less than 100, less than 200, less than 500, or more than 500 nucleotides in length. While

oligonucleotides are often linear, they may, depending on their sequence and conditions, assume a two- or three-dimensional structure. Oligonucleotides may be chemically synthesized by any of a number of methods including sequential synthesis, solid phase synthesis, or any other synthesis method now known or yet to be disclosed. Alternatively, oligonucleotides may be produced by recombinant DNA based methods. One skilled in the art would understand the length of oligonucleotide necessary to perform a particular task. Oligonucleotides may be directly labeled, used as primers in PCR or sequencing reactions, or bound directly to a solid substrate as in oligonucleotide arrays.

A nucleotide is an individual deoxyribonucleotide or ribonucleotide base. Examples of nucleotides include but are not limited to: adenine, thymine, guanine, cytosine, and uracil, which may be abbreviated as A, T, G, C, or U in representations of oligonucleotide or polynucleotide sequence. Any molecule of two or more nucleotide bases, whether DNA or RNA, may be termed a nucleic acid.

An oligonucleotide used to detect to an allele may be affixed to a solid substrate.

Alternatively, the sample may be affixed to a solid substrate and the nucleic acid reagent placed into a mixture. For example, the nucleic acid reagent may be bound to a substrate in the case of an array or the sample may be bound to a substrate as the case of a Southern Blot, Northern blot or other method that affixes the sample to a substrate. A nucleic acid reagent or sample may be covalently bound to the substrate or it may be bound by some non covalent interaction including electrostatic, hydrophobic, hydrogen bonding, Van Der Waals, magnetic, or any other interaction by which an oligonucleotide may be attached to a substrate while maintaining its ability to recognize the allele to which it has specificity. A substrate may be any solid or semi solid material onto which a probe may be affixed, attached or printed, either singly or in the formation of a microarray. Examples of substrate materials include but are not limited to polyvinyl, polysterene, polypropylene, polyester or any other plastic, glass, silicon dioxide or other silanes, hydrogels, gold, platinum, microbeads, micelles and other lipid formations, nitrocellulose, or nylon membranes. The substrate may take any shape, including a spherical bead or flat surface.

A nucleotide is an individual deoxyribonucleotide or ribonucleotide base. Examples of nucleotides include but are not limited to: adenine, thymine, guanine, cytosine, and uracil, which may be abbreviated as A, T, G, C, or U in representations of oligonucleotide or polynucleotide sequence.

In some aspects of the invention, the probe may be affixed to a solid substrate. In other aspects of the invention, the sample may be affixed to a solid substrate. A probe or sample may be covalently bound to the substrate or it may be bound by some non covalent interaction including electrostatic, hydrophobic, hydrogen bonding, Van Der Waals, magnetic, or any other interaction by which a probe such as an oligonucleotide probe may be attached to a substrate while maintaining its ability to recognize the allele to which it has specificity. A substrate may be any solid or semi solid material onto which a probe may be affixed, attached or printed, either singly or in the formation of a microarray. Examples of substrate materials include but are not limited to polyvinyl, polysterene, polypropylene, polyester or any other plastic, glass, silicon dioxide or other silanes, hydrogels, gold, platinum, microbeads, micelles and other lipid formations, nitrocellulose, or nylon membranes. The substrate may take any form, including a spherical bead or flat surface. For example, the probe may be bound to a substrate in the case of an array. The sample may be bound to a substrate as (for example) the case of a Southern Blot, Northern blot or other method that affixes the sample to a substrate.

Kits may also contain reagents that detect proteins, often through the use of an antibody. These kits will contain one or more specific antibodies, buffers, and other reagents configured to detect binding of the antibody to the specific epitope. One or more of the antibodies may be labeled with a fluorescent, enzymatic, magnetic, metallic, chemical, or other label that signifies and/or locates the presence of specifically bound antibody. The kit may also contain one or more secondary antibodies that specifically recognize epitopes on other antibodies. These secondary antibodies may also be labeled. The concept of a secondary antibody also encompasses non- antibody ligands that specifically bind an epitope or label of another antibody. For example, streptavidin or avidin may bind to biotin conjugated to another antibody. Such a kit may also contain enzymatic substrates that change color or some other property in the presence of an enzyme that is conjugated to one or more antibodies included in the kit.

A kit may also contain an indication of a result of the use of the kit that signifies a particular physiological or cellular characteristic. An indication includes any guide to a result that would signal the presence or absence of any physiological or cellular state that the kit is configured to predict. For example, the indication may be expressed numerically, expressed as a color or density of a color, expressed as an intensity of a band, derived from a standard curve, or expressed in comparison to a control. The indication may be communicated through the use of a writing that may be contained physically in or on the kit (on a piece of paper for example), posted on the Internet, mailed to the user separately from the kit, or embedded in a software package. The writing may be in any medium that communicates how the result may be used to predict the cellular or physiological characteristic such as a printed document, a photograph, sound, color, or any combination thereof.

The invention further encompasses pharmaceutical compositions that include the disclosed compound as an ingredient. Such pharmaceutical compositions may take any physical form necessary depending on a number of factors including the desired method of administration and the physicochemical and stereochemical form taken by the disclosed compound or pharmaceutically acceptable salts of the compound. Such physical forms include a solid, liquid, gas, sol, gel, aerosol, or any other physical form now known or yet to be disclosed. The concept of a pharmaceutical composition including the disclosed compound also encompasses the disclosed compound or a pharmaceutically acceptable salt thereof without any other additive. The physical form of the invention may affect the route of administration and one skilled in the art would know to choose a route of administration that takes into consideration both the physical form of the compound and the disorder to be treated. Pharmaceutical compositions that include the disclosed compound may be prepared using methodology well known in the pharmaceutical art. A pharmaceutical composition that includes the disclosed compound may include a second effective compound of a distinct chemical formula from the disclosed compound. This second effective compound may have the same or a similar molecular target as the target or it may act upstream or downstream of the molecular target of the disclosed compound with regard to one or more biochemical pathways.

Pharmaceutical compositions including the disclosed compound include materials capable of modifying the physical form of a dosage unit. In one nonlimiting example, the composition includes a material that forms a coating that holds in the compound. Materials that may be used in such a coating, include, for example, sugar, shellac, gelatin, or any other inert coating agent.

Pharmaceutical compositions including the disclosed compound may be prepared as a gas or aerosol. Aerosols encompass a variety of systems including colloids and pressurized packages. Delivery of a composition in this form may include propulsion of a pharmaceutical composition including the disclosed compound through use of liquefied gas or other compressed gas or by a suitable pump system. Aerosols may be delivered in single phase, bi-phasic, or tri- phasic systems.

In some aspects of the invention, the pharmaceutical composition including the disclosed compound is in the form of a solvate. Such solvates are produced by the dissolution of the disclosed compound in a pharmaceutically acceptable solvent. Pharmaceutically acceptable solvents include any mixtures of more than one solvent. Such solvents may include pyridine, chloroform, propan-l-ol, ethyl oleate, ethyl lactate, ethylene oxide, water, ethanol, and any other solvent that delivers a sufficient quantity of the disclosed compound to treat the affliction without serious complications arising from the use of the solvent in a majority of patients.

Pharmaceutical compositions that include the disclosed compound may also include a pharmaceutically acceptable carrier. Carriers include any substance that may be administered with the disclosed compound with the intended purpose of facilitating, assisting, or helping the administration or other delivery of the compound. Carriers include any liquid, solid, semisolid, gel, aerosol or anything else that may be combined with the disclosed compound to aid in its administration. Examples include diluents, adjuvants, excipients, water, oils (including petroleum, animal, vegetable or synthetic oils.) Such carriers include particulates such as a tablet or powder, liquids such as an oral syrup or injectable liquid, and inhalable aerosols. Further examples include saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, and urea. Such carriers may further include binders such as ethyl cellulose, carboxymethylcellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins; disintegrating agents such as alginic acid, sodium alginate, Primogel, and corn starch; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin, a flavoring agent such as peppermint, methyl salicylate or orange flavoring, or coloring agents. Further examples of carriers include polyethylene glycol, cyclodextrin, oils, or any other similar liquid carrier that may be formulated into a capsule. Still further examples of carriers include sterile diluents such as water for injection, saline solution, physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or digylcerides, polyethylene glycols, glycerin, cyclodextrin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose, thickening agents, lubricating agents, and coloring agents.

The pharmaceutical composition including the disclosed compound may take any of a number of formulations depending on the physicochemical form of the composition and the type of administration. Such forms include solutions, suspensions, emulsions, tablets, pills, pellets, capsules, capsules including liquids, powders, sustained-release formulations, directed release formulations, lyophylates, suppositories, emulsions, aerosols, sprays, granules, powders, syrups, elixirs, or any other formulation now known or yet to be disclosed. Additional examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, hereby incorporated by reference in its entirety.

Methods of administration include, but are not limited to, oral administration and parenteral administration. Parenteral administration includes, but is not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, sublingual, intramsal, intracerebral, iratraventricular, intrathecal, intravaginal, transdermal, rectal, by inhalation, or topically to the ears, nose, eyes, or skin. Other methods of administration include but are not limited to infusion techniques including infusion or bolus injection, by absorption through epithelial or mucocutaneous linings such as oral mucosa, rectal and intestinal mucosa. Compositions for parenteral administration may be enclosed in ampoule, a disposable syringe or a multiple-dose vial made of glass, plastic or other material.

Administration may be systemic or local. Local administration is administration of the disclosed compound to the area in need of treatment. Examples include local infusion during surgery; topical application, by local injection; by a catheter; by a suppository; or by an implant. Administration may be by direct injection at the site (or former site) of a cancer, tumor, or precancerous tissue or into the central nervous system by any suitable route, including intraventricular and intrathecal injection. Intraventricular injection can be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration may be achieved by any of a number of methods known in the art. Examples include use of an inhaler or nebulizer, formulation with an aerosolizing agent, or via perfusion in a fluorocarbon or synthetic pulmonary surfactant. The disclosed compound may be delivered in the context of a vesicle such as a liposome or any other natural or synthetic vesicle.

A pharmaceutical composition formulated so as to be administered by injection may be prepared by dissolving the disclosed compound with water so as to form a solution. In addition, a surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants include any complex capable of non-covalent interaction with the disclosed compound so as to facilitate dissolution or homogeneous suspension of the compound.

Pharmaceutical compositions including the disclosed compound may be prepared in a form that facilitates topical or transdermal administration. Such preparations may be in the form of a liquid solution, cream, paste, lotion, shake lotion, powder, emulsion, ointment, gel base, transdermal patch or iontophoresis device. Examples of bases used in such compositions include opetrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers, thickening agents, or any other suitable base now known or yet to be disclosed.

Addition of a pharmaceutical composition to cancer cells includes all actions by which an effect of the pharmaceutical composition on the cancer cell is realized. The type of addition chosen will depend upon whether the cancer cells are in vivo, ex vivo, or in vitro, the physical or chemical properties of the pharmaceutical composition, and the effect the composition is to have on the cancer cell. Nonlimiting examples of addition include addition of a solution including the pharmaceutical composition to tissue culture media in which in vitro cancer cells are growing; any method by which a pharmaceutical composition may be administered to an animal including intravenous, per os, parenteral, or any other of the methods of administration; or the activation or inhibition of cells that in turn have effects on the cancer cells such as immune cells (e.g. macophages and CD8+ T cells) or endothelial cells that may differentiate into blood vessel structures in the process of angiogenesis or vasculogenesis.

Determination of an effective amount of the disclosed compound is within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. The effective amount of a pharmaceutical composition used to effect a particular purpose as well as a pharmacologically acceptable dose determined by toxicity, excretion, and overall tolerance may be determined in cell cultures or experimental animals by pharmaceutical and toxicological procedures either known now by those skilled in the art or by any similar method yet to be disclosed. One example is the determination of the IC ₅₀ (half maximal inhibitory concentration) of the pharmaceutical composition in vitro in cell lines or target molecules. Another example is the determination of the LD ₅₀ (lethal dose causing death in 50% of the tested animals) of the pharmaceutical composition in experimental animals. The exact techniques used in determining an effective amount will depend on factors such as the type and physical/chemical properties of the pharmaceutical composition, the property being tested, and whether the test is to be performed in vitro or in vivo. The determination of an effective amount of a pharmaceutical composition will be well known to one of skill in the art who will use data obtained from any tests in making that determination. Determination of an effective amount of disclosed compound for addition to a cancer cell also includes the determination of an effective therapeutic amount, including the formulation of an effective dose range for use in vivo, including in humans.

Treatment is contemplated in living entities including but not limited to mammals

(particularly humans) as well as other mammals of economic or social importance, including those of an endangered status. Further examples include livestock or other animals generally bred for human consumption and domesticated companion animals.

The toxicity and therapeutic efficacy of a pharmaceutical composition may be determined by standard pharmaceutical procedures in cell cultures or animals. Examples include the determination of the IC ₅₀ (the half maximal inhibitory concentration) and the LD ₅₀ (lethal dose causing death in 50% of the tested animals) for a subject compound. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The effective amount of the disclosed compound to results in the slowing of expansion of the cancer cells would preferably result in a concentration at or near the target tissue that is effective in slowing cellular expansion in cancer cells, but have minimal effects on non-cancer cells, including non-cancer cells exposed to radiation or recognized chemotherapeutic chemical agents. Concentrations that produce these effects can be determined using, for example, apoptosis markers such as the apoptotic index and/or caspase activities either in vitro or in vivo.

Treatment of a condition is the practice of any method, process, or procedure with the intent of halting, inhibiting, slowing or reversing the progression of a disease, disorder or condition, substantially ameliorating clinical symptoms of a disease disorder or condition, or substantially preventing the appearance of clinical symptoms of a disease, disorder or condition, up to and including returning the diseased entity to its condition prior to the development of the disease.

The addition of a therapeutically effective amount of the disclosed compound

encompasses any method of dosing of a compound. Dosing of the disclosed compound may include single or multiple administrations of any of a number of pharmaceutical compositions that include the disclosed compound as an active ingredient. Examples include a single administration of of a slow release composition, a course of treatment involving several treatments on a regular or irregular basis, multiple administrations for a period of time until a diminution of the disease state is achieved, preventative treatments applied prior to the instigation of symptoms, or any other dosing regimen known in the art or yet to be disclosed that one skilled in the art would recognize as a potentially effective regimen. A final dosing regimen including the regularity of and mode of administration will be dependent on any of a number of factors including but not limited to the subject being treated; the severity of the affliction; the manner of administration, the stage of disease development, the presence of one or more other conditions such as pregnancy, infancy, or the presence of one or more additional diseases; or any other factor now known or yet to be disclosed that affects the choice of the mode of

administration, the dose to be administered and the time period over which the dose is administered.

Pharmaceutical compositions that include the disclosed compound may be administered prior to, concurrently with, or after administration of a second pharmaceutical composition that may or may not include the compound. If the compositions are administered concurrently, they are administered within one minute of each other. If not administered concurrently, the second pharmaceutical composition may be administered a period of one or more minutes, hours, days, weeks, or months before or after the pharmaceutical composition that includes the compound Alternatively, a combination of pharmaceutical compositions may be cyclically administered. Cycling therapy involves the administration of one or more pharmaceutical compositions for a period of time, followed by the administration of one or more different pharmaceutical compositions for a period of time and repeating this sequential administration, in order to reduce the development of resistance to one or more of the compositions, to avoid or reduce the side effects of one or more of the compositions, and/or to improve the efficacy of the treatment.

The invention further encompasses kits that facilitate the administration of the disclosed compound to a diseased entity. An example of such a kit includes one or more unit dosages of the compound. The unit dosage would be enclosed in a preferably sterile container and would be comprised of the disclosed compound and a pharmaceutically acceptable carrier. In another aspect, the unit dosage would comprise one or more lyophilates of the compound. In this aspect of the invention, the kit may include another preferably sterile container enclosing a solution capable of dissolving the lyophilate. However, such a solution need not be included in the kit and may be obtained separately from the lyophilate. In another aspect, the kit may include one or more devices used in administrating the unit dosages or a pharmaceutical composition to be used in combination with the compound. Examples of such devices include, but are not limited to, a syringe, a drip bag, a patch or an enema. In some aspects of the invention, the device comprises the container that encloses the unit dosage.

Pharmaceutical compositions including the disclosed compound may be used in methods of treating cancer. Such methods involve the administration of a therapeutic amount of a pharmaceutical composition that includes the disclosed compound and/or a pharmaceutically acceptable salt thereof to a mammal, preferably a mammal in which a cancer has been diagnosed.

A therapeutic amount further includes the prevention of progression of the cancer to a neoplastic, malignant or metastatic state. Such preventative use is indicated in conditions known or suspected of preceding progression to cancer, in particular, where non- or precancerous cell growth consisting of hyperplasia, metaplasia, or most particularly, dysplasia has occurred (for review of such abnormal growth conditions, see Robbins and Angell, 1976, Basic Pathology, 2d Ed., W. B. Saunders Co., Philadelphia, pp. 68-90, incorporated by reference). Hyperplasia is a form of controlled cell proliferation involving an increase in cell number in a tissue or organ, without significant alteration in structure or activity. For example, endometrial hyperplasia often precedes endometrial cancer and precancerous colon polyps often transform into cancerous lesions. Metaplasia is a form of controlled cell growth in which one type of adult or fully differentiated cell substitutes for another type of adult cell. Metaplasia can occur in epithelial or connective tissue cells. A typical metaplasia involves a somewhat disorderly metaplastic epithelium. Dysplasia is frequently a forerunner of cancer, and is found mainly in the epithelia; it is the most disorderly form of non-neoplastic cell growth, involving a loss in individual cell uniformity and in the architectural orientation of cells. Dysplastic cells often have abnormally large, deeply stained nuclei, and exhibit pleomorphism. Dysplasia characteristically occurs where there exists chronic irritation or inflammation, and is often found in the cervix, respiratory passages, oral cavity, and gall bladder.

Alternatively or in addition to the presence of abnormal cell growth characterized as hyperplasia, metaplasia, or dysplasia, the presence of one or more characteristics of a

transformed phenotype or of a malignant phenotype, displayed in vivo or displayed in vitro by a cell sample derived from a patient can indicate the desirability of prophylactic/therapeutic administration of the pharmaceutical composition that includes the compound. Such

characteristics of a transformed phenotype include morphology changes, looser substratum attachment, loss of contact inhibition, loss of anchorage dependence, protease release, increased sugar transport, decreased serum requirement, expression of fetal antigens, disappearance of the 250,000 dalton cell surface protein, etc. Further examples include leukoplakia, featuring a benign-appearing hyperplastic or dysplastic lesion of the epithelium, or Bowen's disease, a carcinoma in situ. Both of theses are pre-cancerous lesions indicative of the desirability of prophylactic intervention. In another example, fibrocystic disease including cystic hyperplasia, mammary dysplasia, adenosis, or benign epithelial hyperplasia is indicates desirability of prophylactic intervention.

In some aspects of the invention, use of the disclosed compound may be determined by one or more physical factors such as tumor size and grade or one or more molecular markers and/or expression signatures that indicate prognosis and the likely response to treatment with the compound. For example, determination of estrogen (ER) and progesterone (PR) steroid hormone receptor status has become a routine procedure in assessment of breast cancer patients. See, for example, Fitzgibbons et al, Arch. Pathol. Lab. Med. 124:966-78, 2000, incorporated by reference. Tumors that are hormone receptor positive are more likely to respond to hormone therapy and also typically grow less aggressively, thereby resulting in a better prognosis for patients with ER+/PR+ tumors. In a further example, overexpression of human epidermal growth factor receptor 2 (HER-2/neu), a transmembrane tyrosine kinase receptor protein, has been correlated with poor breast cancer prognosis (see, e.g., Ross et al, The Oncologist 8:307-25, 2003), and Her-2 expression levels in breast tumors are used to predict response to the anti-Her-2 monoclonal antibody therapeutic trastuzumab (Herceptin®, Genentech, South San Francisco, CA).

In another aspect of the invention, the diseased entity exhibits one or more predisposing factors for malignancy that may be treated by administration of a pharmaceutical composition including the compound. Such predisposing factors include but are not limited to chromosomal translocations associated with a malignancy such as the Philadelphia chromosome for chronic myelogenous leukemia and t (14 ; 18) for follicular lymphoma; an incidence of polyposis or Gardner's syndrome that are indicative of colon cancer; benign monoclonal gammopathy which is indicative of multiple myeloma, kinship with persons who have had or currently have a cancer or precancerous disease, exposure to carcinogens, or any other predisposing factor that indicates in increased incidence of cancer now known or yet to be disclosed.

The invention further encompasses methods of treating cancer that comprise combination therapies that comprise the administration of a pharmaceutical composition including the disclosed compound and another treatment modality. Such treatment modalities include but are not limited to, radiotherapy, chemotherapy, surgery, immunotherapy, cancer vaccines, radioimmunotherapy, treatment with pharmaceutical compositions other than those which include the disclosed compound, or any other method that effectively treats cancer in

combination with the disclosed compound now known or yet to be disclosed. Combination therapies may act synergistically. That is, the combination of the two therapies is more effective than either therapy administered alone. This results in a situation in which lower dosages of both treatment modality may be used effectively. This in turn reduces the toxicity and side effects, if any, associated with the administration either modality without a reduction in efficacy. In another aspect of the invention, the pharmaceutical composition including the disclosed compound is administered in combination with a therapeutically effective amount of radiotherapy. The radiotherapy may be administered concurrently with, prior to, or following the administration of the pharmaceutical composition including the compound. The radiotherapy may act additively or synergistically with the pharmaceutical composition including the compound. This particular aspect of the invention would be most effective in cancers known to be responsive to radiotherapy. Cancers known to be responsive to radiotherapy include, but are not limited to, Non-Hodgkin's lymphoma, Hodgkin's disease, Ewing's sarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, larynx cancer, cervical cancer, nasopharynx cancer, breast cancer, colon cancer, pancreatic cancer, head and neck cancer, esophogeal cancer, rectal cancer, small-cell lung cancer, non-small cell lung cancer, brain tumors, other CNS neoplasms, or any other such tumor now known or yet to be disclosed.

Examples of pharmaceutical compositions that may be used in combination with the disclosed compound may include nucleic acid binding compositions such as cis- diamminedichloro platinum (II) (cisplatin), doxorubicin, 5-fluorouracil, taxol, and topoisomerase inhibitors such as etoposide, teniposide, irinotecan and topotecan. Still other pharmaceutical compositions include antiemetic compositions such as metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoethanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dimenhydrinate, diphenidol, dolasetron, meclizine, methallatal, metopimazine, nabilone, oxyperndyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiethylperazine, thioproperazine and tropisetron.

Still other examples of pharmaceutical compositions that may be used in combination with the pharmaceutical composition including the disclosed compound are hematopoietic colony stimulating factors. Examples of hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alfa. Alternatively, the pharmaceutical composition including the disclosed compound may be used in combination with an anxiolytic agent. Examples of anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam. Pharmaceutical compositions that may be used in combination with pharmaceutical compositions that include the disclosed compound may include analgesic agents. Such agents may be opioid or non- opioid analgesic. Non-limiting examples of opioid analgesics inlcude morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, normorphine, etorphine, buprenorphine, meperidine, lopermide, anileridine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazocine, pentazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofmac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam, sulindac or any other analgesic now known or yet to be disclosed.

In other aspects of the invention, pharmaceutical compositions including the disclosed compound may be used in combination with a method that involves treatment of cancer ex vivo. One example of such a treatment is an autologous stem cell transplant. In this method, a diseased entity's autologous hematopoietic stem cells are harvested and purged of all cancer cells. A therapeutic amount of a pharmaceutical composition including the disclosed compound may then be administered to the patient prior to restoring the entity's bone marrow by addition of either the patient's own or donor stem cells.

Cancers that may be treated by pharmaceutical compositions including the disclosed compoundeither alone or in combination with another treatment modality include solid tumors such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,

lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, stomach cancer, oral cancer, nasal cancer, throat 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, Wilms'tumor, cervical cancer, uterine cancer, testicular cancer, small cell lung carcinoma, bladder carcinoma, lung cancer, epithelial carcinoma, glioma, glioblastoma multiforme, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, skin cancer, melanoma, neuroblastoma, and retinoblastoma.

Additional cancers that may be treated by pharmaceutical compositions including the disclosed compound include blood borne cancers such as acute lymphoblastic leukemia ("ALL,"), acute lymphoblastic B-cell leukemia, acute lymphoblastic T-cell leukemia, acute myeloblastic leukemia ("AML"), acute promyelocytic leukemia ("APL"), acute monoblastic leukemia, acute erythroleukemic leukemia, acute megakaryoblastic leukemia, acute myelomonocytic leukemia, acute nonlymphocyctic leukemia, acute undifferentiated leukemia, chronic myelocytic leukemia ("CML"), chronic lymphocytic leukemia ("CLL"), hairy cell leukemia, multiple myeloma, lymphoblastic leukemia, myelogenous leukemia, lymphocytic leukemia, myelocytic leukemia, Hodgkin's disease, non-Hodgkin's Lymphoma, Waldenstrom's macroglobulinemia, Heavy chain disease, and Polycythemia vera.

Examples that represent different aspects of the invention follow. Such examples should not be construed as limiting the scope of the disclosure. Alternative mechanistic pathways and analogous structures within the scope of the invention would be apparent to those skilled in the art.

The invention encompasses inhibitors of cell migration activity and inhibitors of effector recruitment activity. Inhibition encompasses any action that hinders, from any detectable level up to and including complete inactivation, the progression of a biological process. Such biological processes include expression of a gene or activities of a gene product, progression of a disease, normal and abnormal metabolic activities, interactions between entities within an organism, or interactions between one organism and another. Further nonlimiting examples of biological processes include development, death, maturation, infection, pain, apoptosis, or homeostasis. Inhibition includes actions that silence or repress the expression of a gene. Inhibition also includes actions that hinder the activity of the R A product, protein product, or

postranslationally modified protein product of a gene. Inhibition may be effectuated through a single agent that inactivates a single gene or gene product, by a single agent that inactivates a combination of more than one gene or gene product, a combination of agents that inactivates a single gene or gene product or a combination of agents that inactivates a combination of more than one gene or gene product.

Inhibition may be effectuated directly by an agent that directly causes the inhibition of a biological process or by agents that trigger one or more different biological processes to effectuate the inhibition of the first biological process. Agents that cause inhibition may also be called inhibitors. Examples of inhibitors include compositions such as compounds that trigger R Ai silencing such as microRNA or siRNA, small molecular compounds, proteins such as soluble receptors or antibodies or any fragment thereof, including an Fab, F(ab) ₂ , Fv, scFv, Fc, phage display antibody, peptibody or any other composition of matter that may inactivate or hinder a biological process. Further nonlimiting examples of inhibitors include X-rays, UV rays, visible light including laser light, and sound.

Cell migration activity includes any mode through which a cell may move in two- dimensional or three-dimensional space. Such migration includes movement through the use of pseudopodia including the adhesion of pseudopodia to a surface, a flagellum, a cilium, acts of amoeboid movement, extravasation, myosin-actin interactions, microtubule extension, or any other process through which a cell moves itself from one place to another or changes its morphology. In one aspect of the invention, cell migration activity is measured through cell adhesion. Using adhesion, cell migration activity may be measured by cell-cell aggregation, monolayer radial migration, including adhesion to a cell matrix comprising laminin, BSA or any other cell matrix component, three dimensional spheroid dispersion, or any other method that measures adhesion based cellular migration in space. Migration activity may be measured by any method that detects that a cell has moved from one place to another or has changed its morphology. Such methods include flow cytometry, capillary electrophoresis, visual examination by light, fluorescence, or electron microscopy, or any such method known in the art or yet to be developed. Inhibitors of cell migration activity are agents that disrupt any molecular or cellular process involved in cell migration activity.

Effector recruitment activity includes any activity of a protein that contributes to the formation of a complex of two or more molecules that serves to catalyze one or more chemical reactions. Effectors include any protein, nucleic acid or other molecule that may be included in a complex that performs one or more biological activities. Recruitment activity encompasses any protein-protein interaction including phosphorylation, dephosphorylation and other enzymatic activities, adhesion, signaling cascades, and cytokine/chemokine interactions, any protein- nucleic acid interactions, such as any of those involved in transcription, translation or DNA replication, or any other process that includes a protein interacting with another molecule.

Inhibitors of effector recruitment activity may disrupt the interaction of a molecule with any of the proteins listed above, the interaction between any of those proteins with each other, and further includes any members of a complex that might be later identified.

In one aspect of the invention, inhibitors of effector recruitment activity may be identified on the basis of their ability to disrupt the binding of a molecule to one or more of its effectors. This specific binding may be measured by any method that allows the measurement of a protein- protein interaction known in the art. Such method include the following examples, alone or in combination as necessary: co-immunoprecipitation, biomolecular fluorescence complementation, fluorescence resonance energy transfer, label transfer, a yeast two-hybrid screen, in- vivo crosslinking, tandem affinity purification, chemical crosslinking, quantitative

immunoprecipitation combined with knock-down (QUICK), dual polarization interferometry, protein-protein docking, static light scattering, immunoprecipitation plus mass-spectrometry, Strep-protein interaction experiment (SPINE), surface plasmon resonance, fluorescence correlation spectroscopy, or any other method of measuring the specific interaction between one protein and another now known in the art or yet to be disclosed.

In another aspect of the invention a glioblastoma patient is treated by first assessing the expression of a target and then treating with an effective dose of an inhibitor of that target, potentially in combination with Temozolimide. The effective dose of a compound is that amount effective to prevent occurrence of the symptoms of a disorder or to treat some symptoms of the disorder from which the patient suffers. Effective dose also includes an effective amount, a therapeutic amount, or any amount sufficient to elicit the desired pharmacological or therapeutic effects, thus resulting in effective prevention or treatment of the disorder. Thus, when treating a patient with glioblastoma, an effective amount of compound is an amount sufficient to slow, or arrest the progression, migration, metastasis, growth, or development of the tumor with the result that life is extended. Prevention includes a delay in onset of symptoms. Treatment includes a decrease in the symptoms associated with the disorder or an amelioration of the recurrence of the symptoms of the disorder. A pharmacologically acceptable dose encompasses any dose that may be administered to a patient that will not be lethal to the patient or cause effects that threaten the health or the life of the patient. Patients include any human being, nonhuman primate, companion animal, or mammal suffering from a disease. In one aspect of the invention, the patient has symptoms that signify the presence of a tumor or other growth in the brain. Such symptoms include headache, seizures, mental or personality changes, mass effect, or one of a number of focal or localized systems including ringing or buzzing sounds, hearing loss, loss of coordination, reduced sensation, weakness or paralysis, difficulty with walking or speech, difficulty keeping balance, decreased muscle control, or double vision. Patients may display one or more different brain tumor types including acoustic neurinoma, astrocytoma, ependyoma, glioblastoma multiforme, meningioma, metastatic tumors originating from another tumor type, mixed glioblastoma,

oligodendroglioblastoma, or pineal region tumor.

EXAMPLE

Members of the TNFR superfamily (TNFRSF), most notably TNFR1, have been shown to play a role in inducing cell invasion and migration in several cancer types. To determine whether other members of the TNFRSF are upregulated in glial tumors, the expression microarray database of 195 clinically annotated brain tumor specimens publicly available at NCBl's Gene Expression Omnibus as dataset GSE4290 was mined. Snap-frozen specimens from epileptogenic foci (NB, n = 24), low-grade astrocytomas (LGA, n=29), and glioblastoma multiforme (GBM, n=82) with clinical information were collected at the Hermelin Brain Tumor Center, Henry Ford Hospital (Detroit, MI) as previously described (See Reference 24). Gene expression profiling was conducted on all samples using Affymetrix U133 Plus 2 GeneChips according to the manufacturer's protocol at the Neuro-Oncology Branch at the National Cancer Institute (Bethesda, MD). For our analysis, gene expression data were normalized in two ways: per chip normalization and per gene normalization across all samples in the collection. For per chip normalization, all expression data on a chip were normalized to the 50th percentile of all values on that chip. For per gene normalization, the data for a given gene were normalized to the median expression level of that gene across all samples. Gene expression differences were deemed statistically significant using parametric tests where variances were not assumed equal (Welch analysis of variance). Expression values were then filtered for highly variable

(differentially expressed) genes (coefficient of variation > 30%) across samples producing a list of 7322 genes. Of the TNFRSF genes analyzed, it was found that TROY/TNFRSF19 expression is significantly differentially expressed among brain specimens. In normal brain specimens, TROY expression is relatively low, but is increased with increasing tumor grade and is significantly higher in GBM samples (n=82) (See Figure 1). To verify the results from the expression profiling, quantitative RT-PCR was performed on independent non-neoplastic (n=10), LGA (n=6), anaplastic astrocytoma (n=4), and GBM (n=22) specimens. Normal brain specimens show relatively low mRNA levels for TROY as compared to the brain tumor samples (p < 0.01). In GBM specimens, the mRNA level of TROY is significantly higher than in normal brain (p < 0.01) (See Figure 2). Next, principal component analysis was done to discern possible relationships between subgroups of samples as in, for example, Reference 34. Kaplan-Meier survival curves were developed for each principal component cluster. One cluster had a median survival time of 401 days (short-term survival, ST) and the other cluster had a median survival time of 952 days (long-term survival, LT). Box-and-whisker plots for TROY expression level in each cluster derived from PC analysis were graphed. Significance between the two populations was tested with a two-sample t-test assuming unequal variances. Analysis of the Affymetrix expression values for TROY in the GBM specimens for each cluster showed that patients with GBM in the short-term survival cluster had higher expression of TROY (10.5) than GBM patients in the long term survival cluster (2.9; /?<0.01) (See Figure 3) This demonstrates that high TROY expression levels correlates with poor patient outcome while low TROY expression level corresponds with good patient outcome.

TROY is expressed in glioblastoma cell lines and siRNA-mediated depletion of TROY suppresses glioblastoma cell migration. The expression of TROY protein was assessed in four different cultured glioblastoma cell lines. The highest level of expression of TROY was seen in Ul 18 cells, the next highest level of expression was seen in U87 cells, and the lowest level of expression was seen in T98G and SNB19 cells (See Figure 4).

RNAi was used to suppress the expression of TROY in each of the four listed glioma cell lines and the migratory behavior of the cells on glioma-derived ECM was examined using a two- dimensional radial cell migration assay (See References 26 and 27). Suppression of TROY protein expression in all glioma cell lines was -80-90% effective with each of two independent siRNA oligonucleotides. Representative results are shown for Ul 18 cells (See Figure 5). Further, suppression of TROY expression by siRNA resulted in a significant (p < 0.05) inhibition of cell migration in all four cell lines (See Figure 18). T98G and SNB19 glioma cell lines that stably express of HA-epitope tagged TROY were produced through lentiviral transduction. These were used to further examine the role TROY signaling in glioma cell migration (See Figure 7). Both the T98G and SNB19 lines normally express low levels of endogenous TROY. The cell lines with HA tagged-TROY showed a ~ 1.8- 2.3-fold increase in cell migration rate (See Figure 8). Migration of the HA-tagged TROY expressing cells was further tested in the context of an authentic brain microenvironment using an ex vivo organotypic rat brain slice model. T98G glioma cells that overexpressed TROY displayed a two-fold increase in the depth of cell invasion after 48 hours relative to controls (See Figure 9). Immuno localization of TROY using an anti-HA antibody revealed that TROY was localized near the cell perimeter and was enriched in lamellipodia (See Figure 10 panel b).

Potential effector molecules of TROY were found through immunoprecipitation experiments coupled with MALDI-TOF MS analysis. In one experiment, T98G cells expressing HA-tagged TROY and control T98G cells transfected with GFP were lysed, immunoprecipitated with anti-HA antibodies, and the immunoprecipitates resolved by SDS-PAGE. Prominent protein bands present in the immunoprecipitates of TROY expressing cells but absent in the

immunoprecipitates of control cells of interest were recovered from the gel. Proteins were eluted, and trypsin-digested. MALDI-TOF and MS-MS analysis of the trypsin digests were performed on a Voyager reflector instrument (Applied Biosystems) and a Q-STAR mass spectrometer (Perceptive Biosystems) in positive ion mode.

The non-receptor protein tyrosine kinase Pyk2 was a candidate sequence identified by mass spectrometry in the TROY immunoprecipitate. Association of TROY with Pyk2 was verified by co-immunoprecipitation. T98G cells transfected with HA-tagged TROY or co- transfected with HA-tagged TROY and Pyk2 were immunoprecipitated with anti-HA antibodies and the precipitates immunoblotted with anti-Pyk2 antibodies (See Figure 11). Both endogenous Pyk2 and transfected Pyk2 co-immunoprecipitated with TROY substantiating the intracellular interaction between TROY and Pyk2.

Depletion of Pyk2 expression by shRNA in TROY overexpressing T98G cells was performed to determine whether the association with Pyk2 was required for TROY-induced stimulation of glioma migration. Suppression of Pyk2 expression by shRNA significantly inhibited TROY stimulated glioma cell migration (See Figure 12). Further, coexpression of a kinase inactive variant of Pyk2 (Pyk2KD) with TROY HA significantly inhibited the migration of the control T98G cells indicating that Pyk2 activity is required for TROY stimulated migration of glioma cells. (See Figure 13). Finally, silencing of Pyk2 expression also inhibited TROY mediated Racl activation (See Figure 15). Together, these results indicate that TROY- mediated glioma cell migration is dependent upon Pyk2 activity.

Rho GTPase family members, particularly Racl (See References 22-24, 28) effect the invasive behavior of glioblastoma cells. As a result, if TROY signaling influences Racl activity, then TROY is a marker of invasive glioblastoma and Racl is an effector molecule of TROY. Ul 18 cells express a high endogenous level of TROY protein expression (See Figure 4) and display high Racl activity (See Figure 14). Reduction of TROY expression in Ul 18 cells by siRNA resulted in decreased activity of Racl (See Figure 14). Further, siRNA mediated reduction of TROY expression induced RhoA activation, showing that TROY signaling modulates Racl and RhoA GTPases activity in opposite directions. Indeed, it has been previously noted that in certain cell types, overexpression of TROY increased RhoA activation (See References 6 and 9) suggesting that TROY signaling may be modulated by cell type specific elements. To validate the effect of TROY on Racl activity, the activation of Racl in glioma cells overexpressing TROY was compared to the activation of Racl in untransfected cells. Overexpression of TROY resulted in a ~ 2-fold induction of Racl activation relative to untransfected cells (See Figure 15).

Since Pyk2 interacts with TROY and mediates TROY-induced migration, the effect of Racl activation induced by TROY expression is dependent upon Pyk2 activity was determined. shRNA-mediated depletion of Pyk2 in TROY overexpressing glioma cells suppressed TROY induced Racl activity to the level of that in control cells. (See Figure 15). This indicates that the TROY-mediated regulation of Racl activation is dependent upon Pyk2. Further, Racl expression in T98G cells overexpressing the TROY receptor was reduced by Racl siRNA. That reduction in Racl expression in was -90% effective in T98G cells and caused a significant inhibition of TROY-mediated cell migration. (See Figure 16).

A recent study suggest that TROY is activated by the TNF family ligand lymphotoxin-a to induce NFKB activation, whereas previous studies have not revealed specific interactions between TROY and any of the TNF family members (See Reference 29). Since Racl can influence multiple downstream signaling pathways, immunoblot analysis of lysates from TROY overexpressing cells were analyzed for detection of various signaling pathways and compared to lysates from untransfected cells. Of the signaling pathways surveyed, increased phosphorylation of Akt, ΙκΒα, and ERKl/2 was seen in TROY overexpressing cells relative to untransfected cells (See Figure 18).

Activation of Akt and NFKB signaling pathways plays a critical role in cell survival. The effect of TROY expression on chemotherapy-induced apoptosis in glioma cells was then determined by comparing, the sensitivity of control Ul 18 glioma cells and Ul 18 glioma cells reduced TROY expression by transfection of TROY specific RNAi to temozolomide treatment. Ul 18 cells with reduced expression of TROY were significantly more sensitive to cell death following temozolomide treatment relative to Ul 18 cells transfected with a negative RNAi control (See Figure 19). Conversely, T98G glioma cells overexpressing TROY were

significantly more resistant to temozolomide induced apoptosis relative to control transfected T98G glioma cells (See Figure 20). Together, these data indicate that TROY stimulated glioma cell migration/invasion increases resistance to chemotherapy-induced cell death in glioma.

A number of RhoGTPases, including Racl Rac3 and Cdc42 (See References 22-25), contribute to glioblastoma cell invasion in vitro. The Rho GTPases are activated by GEFs. There are currently 80 RhoGEFs in the human genome. Of these GEFs, 26 are known Racl activators, and currently, it is not known which Rac GEFs contribute to Racl activity in glial tumors. Rac GEFs that mediate glioma invasion were identified by first mining the NCBI expression microarray database of human brain tumor specimens for the 26 GEFs known to have Rac exchange factor activity. Of the 26 GEFs, Ect2, Trio and Vav3 exhibited increased expression in glioblastomas (GBMs) versus normal brain. Depletion of Ect2, Trio, and Vav3 expression reduced Racl activity in glioblastoma cells which in turn led to a subsequent inhibition of glioblastoma cell migration and invasion. A library of small interfering RNAs (siRNAs) directed against all 26 Rac GEFs in the human genome was used to evaluate the role of Rac in inhibiting glioma invasion in a 96-well format invasion assay. Using this approach, 2 additional Rac GEFs were identified. These two Rac GEF's, Dockl80 and Dock7 contribute to glioma invasion. Knockdown of Dockl80 or Dock7 expression by RNAi significantly reduced glioma invasion in vitro (See Figure 21). Dockl80 has recently been reported (See Reference 30) to be

overexpressed in invasive glioma cells where it regulates Racl activity and glioma cell invasion. To further examine the role of Dock7 in glioblastoma cell invasion using RNAi sequences that inhibit Dock 7 expression to inhibit the migration of SNB19 cells into rat brain slices, a well- established ex vivo organotypic model for glioma invasion. Knockdown of Dock7 expression significantly inhibited invasion relative to control cells (See Figure 22).

A significant limitation of the use of long-term established human glioma cell lines for orthotopic xenografts is their propensity to form discrete, non-invasive tumors with well circumscribed borders that push into the adjacent normal brain tissue (See References 31-33). This is in contrast to the diffuse highly infiltrative growth that defines primary GBM in patients. More important is the loss of genetic features and signatures in long-term established cell lines which are common to primary GBM. A model based on utilizing primary glioma xenografts established and maintained by direct heterotypic transplantation, propagation, and passaging of patient tumor surgical samples in immune deficient mice has been established. Intracranial tumors established with these GBM xenografts retain key histopathological characteristics of the aggressive behavior of the patients' tumors including local invasion at the tumor periphery and invasion along white matter tracks, as well as manifesting key genetic features such as preservation of EGFR amplification status. Therefore, tumors that arise from these xenograft lines adequately model primary GBM in patients (See References 34-36). TROY protein expression was analyzed lysates obtained from 19 xenografts grown orthotopically in murine brain. Examination of TROY expression on the xenograft lysates showed a range of TROY expression. Representative immunoblots showing GBM xenografts with high TROY expression (GBM 10), intermediate levels of TROY expression (GBM6, GBM8), or low levels of TROY expression (GBM44, GBM46) are shown in Figure 23.

Referring now to Figure 1 : TROY mRNA expression levels derived from the NCBI Gene Expression Omnibus GDS1962 dataset are presented as box-and- whisker plots. The box for each gene indicates the interquartile range (25-75th percentile) and the line within this box indicates the median value. Bottom and top bars of the whisker indicate the 10th and 90th percentiles, respectively. Outliers are represented by closed circles. Significance between the indicated classes of brain specimens was tested using a two-sample t test assuming unequal variances. (NB = non-neoplastic brain; OL, Oligodendrogliomas; Astro = low grade astrocytomas; GBM = glioblastoma multiforme). Referring now to Figure 2 Quantitative real-time PCR analysis of TROY expression in non-neoplastic brain (NB), grade 1 low grade astrocytoma (LGA), grade 2- 3 Astrocytomas (Astro) and glioblastoma multiforme (GBM) indicates that a higher level of

TROY expression signifies increased tumor grade. Values were normalized to histone H3.3 and HPRT1 reference genes. Data are presented as box-and- whisker plots. Referring now to Figure 3: principal component analysis of brain tumors from NCBI Gene Expression Omnibus

GDS1962 dataset revealed two groups differing by their survival and were denoted as long term (LT) survival and short-term (ST) survival. These indicate that a higher level of TROY expression signifies an association with short-term survival. Box-and-whisker plots for TROY expression in GBM specimens for each cluster are shown. Significance between the two populations was tested with a two-sample t test assuming unequal variances.

Referring now to Figure 4: T98G, SNB19, U87, and Ul 18 cell lysates were analyzed for endogenous level of TROY expression by immunoblotting. All express TROY with U87 and Ul 18 cells having the highest expression level. The levels of a-tubulin protein were also immunob lotted to ensure equal sample loading. Referring now to Figure 5: knockdown of TROY expression in Ul 18 cells by two independent siRNA oligonucleotides. Note reduced expression of TROY protein in TROY-1 and TROY-2 transfected Ul 18 cells. Referring now to Figure 18: the migration rate of each of the four cell lines was slowed when the cell lines were transfected with siRNA oligonucleotides targeting TROY. siRNA targeting luciferase waas used as a negative control. Migration rate was determined after 24 h migration on glioma derived ECM (*- p < 0.05).

Referring now to Figure 7: Lysates of T98G or SNB19 cells transduced with empty lentiviral vector (v) or lentiviral vector encoding HA-epitope tagged TROY immunoblotted with anti-HA antibody shows that TROY is overexpressed in cell lines transfected with the TROY construct. Referring now to Figure 8: the migration rate of HA-TROY expressing glioma cells is faster than that of cells transduced with a negative control construct. Cell migration was assessed over 48 h. Data represents the average of three independent experiments (*, p < 0.01; **, p < 0.05). Referring now to Figure 9: T98G cells stably expressing green fluorescent protein were transduced with lentiviruses expressing HA-tagged TROY. Cells were implanted into the bilateral putamen on rat organotypic brain slices and observed at 48 h. Depth of invasion was calculated from Z-axis images collected by confocal laser scanning microscopy. The mean value of the depth of invasion was obtained from six independent experiments (*, p < 0.01). Cell lines transduced with a construct containing TROY displayed significantly greater depth of invasion. Referring now to Figure 10: immunofluorescent staining for Troy in T98G-Troy-HA cells using an anti-HA antibody shows that TROY localizes at the membrane periphery and within cellular extensions.

Referring now to Figure 11 : The top panel indicates that lysates of negative control transfected T98G cells, T98G cells transfected with HA-TROY, T98G cells transfected with Pyk2, or T98G cells cotransfected with HA-TROY and Pyk2 show expression of the transfected constructs when immunoblotted with anti-Pyk2 or anti-HA antibodies. In the bottom panel, the same cell lines were immunoprecipitated with anti-HA antibody and the precipitates

immunoblotted with anti-HA or anti-Pyk2 indicating that Pyk2 associates with TROY. Referring now to Figure 12: The inhibition of Pyk2 expression by RNAi targeting Pyk2 suppresses Troy- induced glioma migration. Migration rate of T98G, T98G-Troy-HA, and T98G-Troy-HA cells transfected with a shRNA targeting Pyk2 was assessed over 24 h using a radial migration assay on 10 μg/ml laminin substrate (*, p < 0.01). T98G cells overexpressing TROY migrate at a faster rate than T98G cells that lack the TROY expressing construct. This effect is negatived by transfection with Pyk2-specific shRNA. Referring now to Figure 13: inhibition of Pyk2 activity inhibits Troy-induced glioma migration. T98G or T98G-Troy-HA expressing cells were infected with recombinant adenoviruses expressing a Pyk2 variant lacking the Pyk2 kinase domain.

(Pyk2KD). Cell migration was assessed over 24 h using a radial migration assay on 10 μg/ml laminin substrate (*, p < 0.01). Transfection of the Pyk2KD construct into T98G cells that do not overexpress TROY slowed the migration rate of those cells. T98G cells overexpresing TROY migrate at an even faster rate, but this effect is mitigated by transfection with Pyk2KD.

Referring now to Figure 14: Ul 18 cells were left untransfected (NT), transfected with an siRNA targeting nonmammalian luciferase (ctrl), or an siRNA targeting TROY (Troy-1). Cells were cultured under serum- free medium for an additional 16 fir prior to RhoA and Racl activation assays. Immunoblots show that RhoA is more likely to be phosphorylated and Rac-1 is dephosphorylated upon suppression of TROY 1 expression. Referring now to Figure 15: T98G and T98G-Troy-HA cells were transfected with a shRNA targeting Pyk2 and cultured under serum- free medium for 16 h. Lysates were then analyzed for activation of Racl . Overexpression of TROY leads to more Racl phosphorylation. This effect is diminished when Pyk2 expression is suppressed. Referring now to Figure 16: suppression of Racl expression by siRNA suppresses Troy-induced glioma cell migration. T98G and T98G-TROY-HA cells were transfected with an siRNA oligonucleotide targeting Racl . Cell migration was assessed over 24 h using a radial migration assay on 10 mg/ml laminin substrate (*, p < 0.01). Suppression of Racl expression reduced the migration rate of the cells indicating that TROY-1 mediated migration works through Racl . Referring now to Figure 17: a Western blot validating suppression of Racl and HA-TROY expression was performed.

Referring now to Figure 18: TROY overexpression induces activation of Akt, NFkB and Erkl/2 signaling pathways. Cellular lysates of T98G glioma cells or T98G cells overexpressmg TROY (left panel) and SNB19 glioma cells or SNB19 cells overexpressmg TROY (right panel) were immunob lotted with the indicated antibodies. Equal sample loading was verified by immunoblotting lysates with an anti-a-tubulin antibody. Western blots indicate phosphorylation of Akt, IkBa, and Erkl/2.

Referring now to Figure 19: Ul 18 cells were transfected with a siRNA targeting TROY (Troy-1) or a siRNA targeting a nonmammalian gene. Cells were then treated with 250 μΜ of temozolomide (TMZ) or vehicle (DMSO) for 48 h. The percentage of cell viability was measured by Alamar Blue assay and normalized to the control siRNA untreated with TMZ (*, p < 0.01; **, p < 0.001). The results indicate that when TROY expression is suppressed, the cells are rendered sensitive to temozolimide. Referring now to Figure 20: T98G and T98G expressing TROY-HA were treated with 250 μΜ of TMZ or vehicle (DMSO) for 48 h. The percentage of cellular apoptosis was measured by annexin V staining followed by flow cytometry. Data represents the mean and S.D. from three independent experiments with each experiment conducted in triplicate (**, p < 0.001). The results show that overexpression of TROY decreases the number of cells that apoptose upon treatment with temozolimide and that overexpression of TROY increases resistance to temozolimide.

Five known Racl activators were assessed for their ability to contribute to glioma invasion identified in a focused RhoGEF genome-wide siRNA screening approach. Referring now to Figure 21 : SNB19 cells transfected with siRNAs targeting luciferase (ctrl), Dockl80, or Dock7 were plated in transwell invasion chambers coated with Matrigel, and 24 h later, cells that had migrated through the filter were stained and counted. Shown are the mean of at least two independent experiments; bars, ±SE (*, p < 0.001). Results indicate that Dockl80 and Dock7 are implicated in the invasive phenotype of glioma cells. Referring now to Figure 22: SNB19-GFP cells were transfected with a siRNA targeting luciferase (ctrl) or two independent siRNAs targeting Dock7. After 24 h, cells were implanted into the bilateral putamen on rat organotypic brain slices and cultured for 48 h. Depth of invasion was calculated from Z-axis images collected by confocal laser scanning microscopy. The mean value of the depth of invasion (+/- SEM) was obtained from three independent experiments, performed in triplicates (*, p < 0.01). The results further indicate a role of Dock7 in the development of an invasive phenotype.

Referring now to Figure 23 : Total cellular lystates from glioblastoma multiforme xenografts grown in murine brain orthopically were collected and immunoblotted for human TROY. Ponceau staining was used as a loading control.

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