WO2018094325A1

WO2018094325A1 - Therapeutic modulation of oncogenes by pharmacologic top2 targeting for cancer

Info

Publication number: WO2018094325A1
Application number: PCT/US2017/062543
Authority: WO
Inventors: Adam Mendel Sonabend WORTHALTER; Eric FELDSTEIN; Edgar F. GONZALEZ-BUENDIA
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2016-11-18
Filing date: 2017-11-20
Publication date: 2018-05-24

Abstract

The present invention relates to inhibiting topoisomerases as a target for downregulating oncogene expression and inhibiting tumor growth. In certain embodiments, methods for treating glioma using a TOP2 inhibitor are provided. In additional embodiments, two biomarkers have been identified for use in determining tumor susceptibility to TOP2 inhibitor treatment.

Description

THERAPEUTIC MODULATION OF ONCOGENES BY PHARMACOLOGIC

TOP2 TARGETING FOR CANCER

Cross Reference to Related Application

This application claims priority to U.S. Provisional Application Number 62/423,869 filed on November 18, 2016, the content of which is incorporated by reference herein in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under grant DP5OD021356 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to inhibiting topoisomerases as a target for downregulating oncogene expression and inhibiting tumor growth. In certain embodiments, methods for inhibiting PDGFRA and MYC oncogenes in a glioma tumor, comprising contacting the glioma tumor with an effective amount of a topoisomerase inhibitor are provided.

BACKGROUND OF THE INVENTION

Gliomas are brain tumors with a remarkable heterogeneous gene expression pattern. Epigenetic factors contribute to the regulation of these transcriptional profiles, yet these mechanisms are not well understood. Deactivating multiple oncogenes as a means of controlling glioma growth is a large challenge. A series of therapeutics have been created to inhibit Tyrosine kinase activity related to the oncogenic growth- promoting phosphorylation pathway from growth factor cell receptors such as EGFR, and PDGFRA, among others. These kinase inhibitors are being tested in clinical trials, but it is known that tumor cells can overcome these pathway inhibitions as there is redundancy. Gliomas have important changes in gene expression that lead to up-regulation of several oncogenes. Whereas these oncogenes are key for tumor growth, therapies to target transcription of specific genes are scant to none. Our approach allows targeting the expression of 2 ongocenes using drugs that are currently available. Thus, there is a large unmet need for new cancer therapeutics, and in particular for therapeutics that are effective against brain tumors, and in particular against gliomas. SUMMARY OF THE INVENTION

In certain embodiments, the present invention relates to a method for inhibiting tumor growth in a patient in need thereof, comprising administering an effective amount of a topoisomerase inhibitor to the patient.

In certain embodiments, the tumor comprises a brain tumor. In additional embodiments, the brain tumor is a glioma.

In certain embodiments, the present invention relates to a method for inhibiting PDGFRA and MYC oncogenes in a glioma tumor, comprising contacting the glioma tumor with an effective amount of a topoisomerase inhibitor.

In certain embodiments, the topoisomerase inhibitor comprises a TOP2 inhibitor.

In additional embodiments, the TOP2 inhibitor comprises TOP2A/B. In yet additional embodiments, TOP2A/B inhibitor comprises ICFR-193.

In certain embodiments, the TOP2A/B inhibitor comprises ICFR-193, MST-16,

ICFR-154, razoxane, or any combination thereof.

In certain embodiments, prior to administering treatment, the tumor is tested for PDGFRA expression, MYC expression, or any combination thereof, wherein overexpression of PDGFRA, MYC, or a combination thereof, above a baseline or reference level indicates a tumor likely to be sensitive to TOP2 inhibitor treatment, and in such instances administering an effective amount of a topoisomerase inhibitor to the patient.

In certain embodiments, the method further comprises determining expression levels in the tumor of one or any combination of PDGFRA expression, MYC expression, or any combination thereof, wherein overexpression of PDGFRA, MYC, or a combination thereof, above a baseline or reference level indicates a tumor likely to be sensitive to TOP2 inhibitor treatment; and in such instances administering an effective amount of a topoisomerase inhibitor to the patient. BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-B: Fig. 1A is graphic expression array data from human glioblastomas showing elevated expression of TOP2A and B in the proneural group (TCGA). The mesenchymal group have the lowest expression for TOP2A and B. The values for TOP2A% are shown as the left bar for each set; while the TOP2B% is shown as the right bar of each set. Fig. IB is a heatmap showing expression of genes identified as having TOP2B activity on promoters (defined as having binding on ChIP— seq) in the presence of TOP2 inhibitor ICRF193 or following treatment with DMSO (control). Of note, PDGFRA and MYC are down-regulated by TOP2 inhibition.

Figures 2A-C: Results showing TOP2A and TOP2B binding to PDGFRA gene in some glioma cell lines (A). Close up view of TOP2B binding to PDGFRA promoter and gene body determined by ChlP-seq (Fig. 2B left) and that this binding is associated with elevated expression across 4 human glioma cell lines (Fig. 2B right). Validation of these findings was done by ChlP-PCR (Fig. 2C).

Figures 3A-B are results showing TOP2A and TOP2B binding to MYC gene in some glioma cell lines (Fig. 3A). Close up view of TOP2B binding to MYC promoter and gene body determined by ChlP-seq (Fig. 3B left) and that this binding is associated with elevated expression across 4 human glioma cell lines (Fig. 3B right).

Figure 4. Gene expression data from human glioma patients (Gliovis, TCGA data) shows a correlation between TOP2B expression with PDGFRA and MYC.

Figure 5. Decatenation assay showing that a dose of 10 μΜ of TOP2 inhibitor ICRF193 significantly decreased the enzymatic activity of TOP2 on glioma cell line TS543.

Figures 6A-B. ChIP PCR showing no TOP2A/B binding to tumor-suppressor gene Rbl (Fig. 6A). TOP2 inhibition with ICRF193 had no significant effect on expression of oncogenes Rbl or p53 on multiple cell lines (Fig. 6B).

Figure 7. TOP2 inhibition led to down-regulation of the mRNA (top) and protein (bottom) of PDGFRA (left) and MYC (right). Transcript levels were determined by RNA-seq, and protein by Western Blot. Figure 8. TOP2 inhibition led to impairment of MYC activity demonstrated as down-regulation of MYC targets in glioma cells TS543 and BT142. Transcript levels were determined by RNA-seq. Analysis of expression was GSVA.

Figure 9. Treatment of human glioma cell lines with ICRF193 and the TOP2 inhibitor drug MST16 leads to significant down-regulation of PDGFRA and MYC determined by qRT-PCR.

Figure 10. Decrease in cell viability elicited by two separate TOP2 inhibitors is highly correlated, as cell viability following treatment with TOP2 inhibitors is related to on-target effect.

Figure 11. Correlation between expression (RFPKM from RNA-seq data) of

PDGFRA and MYC with susceptibility to TOP2 inhibitor MST-16 (determined by dose response area under the curve [AUC]).

Figure 12. Validation of finding on an independent dataset of patient-derived glioma xenograft cell lines. Correlation between expression (RFPKM counts) of PDGFRA with susceptibility to TOP2 inhibitor MST-16 (determined by dose response area under the curve [AUC]).

DETAILED DESCRIPTION

The present invention is based at least in part, upon targeting multiple oncogenes simultaneously by greatly down-regulating their expression. In certain embodiments, the present invention relates to a method for inhibiting tumor growth in a patient in need thereof, comprising administering an effective amount of a topoisomerase inhibitor to the patient. The present invention also includes methods for inhibiting PDGFRA and MYC oncogenes in a glioma tumor, comprising contacting the glioma tumor with an effective amount of a topoisomerase inhibitor.

TOP2A/B are helicases implicated in transcriptional regulation in other contexts, and regulate transcription by inducing double- strand DNA breaks enhancing chromatin accessibility. The gene sequence for TOP2A can be found at Gene ID:7153. TOP2B (UniProtKB-Q02880, human) has been shown to interact with CTCF, a transcription factor capable of binding heterochromatin. (See, Tiwari et ah, PNAS 2012; 109(16):E934-43; and Thakurela et al, Nature Comm 2013; 4:2478.). While there have been general studies evaluating the role of TOP2 in epigenetic modulation of transcription of specific genes, these studies have been limited to stem cells and neurons.

The present results show that TOP2A/B regulate the transcription of oncogenes in specific subsets of gliomas. Based on this hypothesis, it is expected that CTCF plays a role in recruiting TOP2A/B to these loci.

Furthermore, the present results indicate that in certain instances, patients diagnosed with glioma and who also exhibit overexpression of PDGFRA or MYC, or any combination thereof in the glioma tumor tissue, will be likely to exhibit susceptibility to a TOP2 inhibitor, such as, for example MST- 16. For example, the top ranking tumors in Fig. 11 and 12 are susceptible to treatment with a TOP2 inhibitor, while the lower ranking ones are not be susceptible to treatment with a TOP2 inhibitor.

Definitions

So that the invention may be more readily understood, certain technical and scientific terms are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

"Activation," "stimulation," and "treatment," as it applies to cells or to receptors, may have the same meaning, e.g., activation, stimulation, or treatment of a cell or receptor with a ligand, unless indicated otherwise by the context or explicitly. "Ligand" encompasses natural and synthetic ligands, e.g., cytokines, cytokine variants, analogues, muteins, and binding compounds derived from antibodies. "Ligand" also encompasses small molecules, e.g., peptide mimetics of cytokines and peptide mimetics of antibodies. "Activation" can refer to cell activation as regulated by internal mechanisms as well as by external or environmental factors. "Response," e.g., of a cell, tissue, organ, or organism, encompasses a change in biochemical or physiological behavior, e.g., concentration, density, adhesion, or migration within a biological compartment, rate of gene expression, or state of differentiation, where the change is correlated with activation, stimulation, or treatment, or with internal mechanisms such as genetic programming.

"Activity" of a molecule may describe or refer to the binding of the molecule to a ligand or to a receptor, to catalytic activity; to the ability to stimulate gene expression or cell signaling, differentiation, or maturation; to antigenic activity, to the modulation of activities of other molecules, and the like. "Activity" of a molecule may also refer to activity in modulating or maintaining cell-to-cell interactions, e.g., adhesion, or activity in maintaining a structure of a cell, e.g., cell membranes or cytoskeleton. "Activity" can also mean specific activity, e.g., [catalytic activity]/[mg protein], or [immunological activity]/[mg protein], concentration in a biological compartment, or the like. "Activity" may refer to modulation of components of the innate or the adaptive immune systems.

"Administration" and "treatment," as it applies to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refers to contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. "Administration" and "treatment" also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell. The term "subject" includes any organism, preferably an animal, more preferably a mammal {e.g., rat, mouse, dog, cat, rabbit) and most preferably a human, including a human patient.

"Treat" or "treating" refers to administering a therapeutic agent, such as a composition containing any of the present TOP2 inhibitors, or similar compositions described herein, internally or externally to a subject or patient having one or more disease symptoms, or being suspected of having a disease or being at elevated at risk of acquiring a disease, for which the agent has therapeutic activity. Typically, the agent is administered in an amount effective to alleviate one or more disease symptoms in the treated subject or population, whether by inducing the regression of or inhibiting the progression of such symptom(s) by any clinically measurable degree. The amount of a therapeutic agent that is effective to alleviate any particular disease symptom (also referred to as the "therapeutically effective amount") may vary according to factors such as the disease state, age, and weight of the patient, and the ability of the drug to elicit a desired response in the subject Whether a disease symptom has been alleviated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom. While an embodiment of the present invention (e.g., a treatment method or article of manufacture) may not be effective in alleviating the target disease symptom(s) in every subject, it should alleviate the target disease symptom(s) in a statistically significant number of subjects as determined by any statistical test known in the art such as the Student's t-test, the chi²-test, the U-test according to Mann and Whitney, the Kruskal- Wallis test (H-test), Jonckheere-Terpstra-test and the Wilcoxon-test.

"Treatment," as it applies to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, to research and diagnostic applications.

TOP2 Inhibitors (topoisomerase II inhibitors). These inhibitors target the N- terminal ATPase domain of topo II and prevent topo II from turning over. Examples of topoisomerase inhibitors include: ICRF-193 (Robinson, Helen; Bratlie-Thoresen, Sigrid; Brown, Robert; Gillespie, David A.F. (2007). "Chkl is required for G2/M Checkpoint Response Induced by the Catalytic Topoisomerase II Inhibitor ICRF-193". Cell Cycle. 6 (10): 1265-7.). The structure of this compound bound to the ATPase domain was solved by Classen (Proceedings of the National Academy of Sciences, 2004) showing that the drug binds in a non-competitive manner and locks down the dimerization of the ATPase domain (Baird, C. L.; Gordon, MS; Andrenyak, DM; Marecek, JF; Lindsley, JE (2001). "The ATPase Reaction Cycle of Yeast DNA Topoisomerase II. SLOW RATES OF ATP RESYNTHESIS AND Pi RELEASE". Journal of Biological Chemistry. 276 (30): 27893- 8.). Several classes of compounds have been described that target TOP2 without stabilizing TOP2 covalent complexes. The most important class of compounds, the bisdioxopiperazines, include ICRF-159, ICRF-187 and MST-16. These drugs have two activities: they are potent chelating agents and they block TOP2 in the catalytic cycle after strand passage but before the hydrolysis of the second ATP (See John L. Nitiss; Nat Rev Cancer: 2009 May ; 9(5): 338-350). Another example of a TOP2 inhibitor is genistein. Thus, in certain embodiments, it is expected that any TOP2 inhibitor would work in the susceptible patient population identified herein.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that not all progeny will have precisely identical DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

With respect to cells, the term "isolated" refers to a cell that has been isolated from its natural environment (e.g., from a tissue or subject). The term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes can occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants. As used herein, the terms "recombinant cell" refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced.

Pharmaceutical Compositions and Administration

To prepare pharmaceutical or sterile compositions of the compositions of the present invention, the present compound may be admixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

Formulations of therapeutic and diagnostic agents may be prepared by mixing with acceptable carriers, excipients, or stabilizers in the form of, e.g., lyophilized powders, slurries, aqueous solutions or suspensions (see, e.g., Hardman, et al. (2001) Goodman and Gilman 's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

Toxicity and therapeutic efficacy of the therapeutic compositions, administered alone or in combination with another agent, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index (LD50/ ED50). In particular aspects, therapeutic compositions exhibiting high therapeutic indices are desirable. 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 of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration.

In an embodiment of the invention, a composition of the invention is administered to a subject in accordance with the Physicians' Desk Reference 2003 (Thomson

Healthcare; 57th edition (November 1, 2002)).

The mode of administration can vary. Suitable routes of administration include intranasal, nasal, oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intra- arterial.

In particular embodiments, the composition or therapeutic can be administered by an invasive route such as by injection (see above). In further embodiments of the invention, the composition, therapeutic, or pharmaceutical composition thereof, is administered intravenously, subcutaneously, intramuscularly, intraarterially, intra- articularly (e.g. in arthritis joints), intratumorally, or by inhalation, aerosol delivery.

Administration by non-invasive routes {e.g., orally; for example, in a pill, capsule or tablet) is also within the scope of the present invention. Compositions can be administered with medical devices known in the art. For example, a pharmaceutical composition of the invention can be administered by injection with a hypodermic needle, including, e.g., a prefilled syringe or autoinjector.

The pharmaceutical compositions of the invention may also be administered with a needleless hypodermic injection device; such as the devices disclosed in U.S. Patent Nos. 6,620, 135; 6,096,002; 5,399, 163; 5,383,851 ; 5,312,335; 5,064,413; 4,941,880; 4,790,824 or 4,596,556.

Alternately, one may administer the present compound or other TOP2 inhibitors, or related compound in a local rather than systemic manner, for example, via injection of directly into the desired target site, often in a depot or sustained release formulation.

Determination of the appropriate dose is made by the clinician, e.g. , using parameters or factors known or suspected in the art to affect treatment. Generally, the dose begins with an amount somewhat less than the optimum dose and it is increased by small increments thereafter until the desired or optimum effect is achieved relative to any negative side effects. Important diagnostic measures include those of symptoms of, e.g. , the inflammation or level of inflammatory cytokines produced. In general, it is desirable that a biologic that will be used is derived from the same species as the animal targeted for treatment, thereby minimizing any immune response to the reagent.

As used herein, "inhibit" or "treat" or "treatment" includes a postponement of development of the symptoms associated with a disorder and/or a reduction in the severity of the symptoms of such disorder. The terms further include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result has been conferred on a vertebrate subject with a disorder, disease or symptom, or with the potential to develop such a disorder, disease or symptom.

As used herein, the terms "therapeutically effective amount", "therapeutically effective dose" and "effective amount" refer to an amount of the present compound or other TOP2 inhibitors or inhibitor compound of the invention that, when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to cause a measurable improvement in one or more symptoms of a disease or condition or the progression of such disease or condition. A therapeutically effective dose further refers to that amount of the compound sufficient to result in at least partial amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. When applied to an individual active ingredient administered alone, a therapeutically effective dose refers to that ingredient alone. When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously. An effective amount of a therapeutic will result in an improvement of a diagnostic measure or parameter by at least 10%; usually by at least 20%; preferably at least about 30%; more preferably at least 40%, and most preferably by at least 50%. An effective amount can also result in an improvement in a subjective measure in cases where subjective measures are used to assess disease severity.

The terms "sample" or "biological sample" as used herein, refers to a sample of biological fluid, tissue, or cells, in a healthy and/or pathological state obtained from a subject. Such samples include, but are not limited to, blood, bronchial lavage fluid, sputum, saliva, urine, lymph fluid, tissue or fine needle biopsy samples, peritoneal fluid, cerebrospinal fluid, and includes supernatant from cell lysates, lysed cells, cellular extracts, and nuclear extracts. In some embodiments, the whole blood sample is further processed into serum or plasma samples. In some embodiments, the sample includes blood spotting tests.

The terms "identification", "identify", "identifying" and the like as used herein means to recognize a disease state or a clinical manifestation or severity of a disease state in a subject or patient. The term also is used in relation to test agents and their ability to have a particular action or efficacy. In certain preferred embodiments, identification refers to the ability to distinguish cell lines or eventually patients that are susceptible to TOP2 inhibitor therapy.

The terms "prediction", "predict", "predicting" and the like as used herein means to tell in advance based upon special knowledge. The term "agent" as used herein means a substance that produces or is capable of producing an effect and would include, but is not limited to, chemicals, pharmaceuticals, biologies, small organic molecules, antibodies, nucleic acids, peptides, and proteins.

The phrase "therapeutically effective amount" is used herein to mean an amount sufficient to cause an improvement in a clinically significant condition in the subject, or delays or minimizes or mitigates one or more symptoms associated with the disease, or results in a desired beneficial change of physiology in the subject.

As used herein, the term "isolated" and the like means that the referenced material is free of components found in the natural environment in which the material is normally found. In particular, isolated biological material is free of cellular components. In the case of nucleic acid molecules, an isolated nucleic acid includes a PCR product, an isolated mRNA, a cDNA, an isolated genomic DNA, or a restriction fragment. In another embodiment, an isolated nucleic acid is preferably excised from the chromosome in which it may be found. Isolated nucleic acid molecules can be inserted into plasmids, cosmids, artificial chromosomes, and the like. Thus, in a specific embodiment, a recombinant nucleic acid is an isolated nucleic acid. An isolated protein may be associated with other proteins or nucleic acids, or both, with which it associates in the cell, or with cellular membranes if it is a membrane-associated protein. An isolated material may be, but need not be, purified.

The term "purified" and the like as used herein refers to material that has been isolated under conditions that reduce or eliminate unrelated materials, i.e., contaminants. For example, a purified protein is preferably substantially free of other proteins or nucleic acids with which it is associated in a cell; a purified nucleic acid molecule is preferably substantially free of proteins or other unrelated nucleic acid molecules with which it can be found within a cell. As used herein, the term "substantially free" is used operationally, in the context of analytical testing of the material. Preferably, purified material substantially free of contaminants is at least 50% pure; more preferably, at least 90% pure, and more preferably still at least 99% pure. Purity can be evaluated by chromatography, gel electrophoresis, immunoassay, composition analysis, biological assay, and other methods known in the art. The terms "expression profile" or "gene expression profile" refers to any description or measurement of one or more of the genes that are expressed by a cell, tissue, or organism under or in response to a particular condition. Expression profiles can identify genes that are up-regulated, down-regulated, or unaffected under particular conditions. Gene expression can be detected at the nucleic acid level or at the protein level. The expression profiling at the nucleic acid level can be accomplished using any available technology to measure gene transcript levels. For example, the method could employ in situ hybridization, Northern hybridization or hybridization to a nucleic acid microarray, such as an oligonucleotide microarray, or a cDNA microarray. Alternatively, the method could employ reverse transcriptase-polymerase chain reaction (RT-PCR) such as fluorescent dye-based quantitative real time PCR (TaqMan® PCR). In the Examples section provided below, nucleic acid expression profiles were obtained using Affymetrix GeneChip® oligonucleotide microarrays. The expression profiling at the protein level can be accomplished using any available technology to measure protein levels, e.g., using peptide- specific capture agent arrays.

The terms "gene signature" and "signature genes" will be used interchangeably herein and mean the particular transcripts that have been found to be differentially expressed in certain subpopulations of cancer patients, as described herein. For example, those differentially expressed oncogenes including MYC and PDGFRA. It is noted that differential levels of the corresponding proteins, will also be useful as a marker or signature for stratifying, prognosing or diagnosing patients as described herein.

The terms "gene", "gene transcript", and "transcript" are used somewhat interchangeable in the application. The term "gene", also called a "structural gene" means a DNA sequence that codes for or corresponds to a particular sequence of amino acids which comprise all or part of one or more proteins or enzymes, and may or may not include regulatory DNA sequences, such as promoter sequences, which determine for example the conditions under which the gene is expressed. Some genes, which are not structural genes, may be transcribed from DNA to RNA, but are not translated into an amino acid sequence. Other genes may function as regulators of structural genes or as regulators of DNA transcription. "Transcript" or "gene transcript" is a sequence of RNA produced by transcription of a particular gene. Thus, the expression of the gene can be measured via the transcript.

The term "antisense DNA" is the non-coding strand complementary to the coding strand in double-stranded DNA.

The term "genomic DNA" as used herein means all DNA from a subject including coding and non-coding DNA, and DNA contained in introns and exons.

The term "nucleic acid hybridization" refers to anti-parallel hydrogen bonding between two single-stranded nucleic acids, in which A pairs with T (or U if an RNA nucleic acid) and C pairs with G. Nucleic acid molecules are "hybridizable" to each other when at least one strand of one nucleic acid molecule can form hydrogen bonds with the complementary bases of another nucleic acid molecule under defined stringency conditions. Stringency of hybridization is determined, e.g., by (i) the temperature at which hybridization and/or washing is performed, and (ii) the ionic strength and (iii) concentration of denaturants such as formamide of the hybridization and washing solutions, as well as other parameters. Hybridization requires that the two strands contain substantially complementary sequences. Depending on the stringency of hybridization, however, some degree of mismatches may be tolerated. Under "low stringency" conditions, a greater percentage of mismatches are tolerable (i.e., will not prevent formation of an anti-parallel hybrid).

The terms "vector", "cloning vector" and "expression vector" mean the vehicle by which a DNA or RNA sequence (e.g. a foreign gene) can be introduced into a host cell, so as to transform the host and promote expression ( e.g. transcription and translation) of the introduced sequence. Vectors include, but are not limited to, plasmids, phages, and viruses. Vectors typically comprise the DNA of a transmissible agent, into which foreign DNA is inserted. A common way to insert one segment of DNA into another segment of DNA involves the use of enzymes called restriction enzymes that cleave DNA at specific sites (specific groups of nucleotides) called restriction sites. A "cassette" refers to a DNA coding sequence or segment of DNA which codes for an expression product that can be inserted into a vector at defined restriction sites. The cassette restriction sites are designed to ensure insertion of the cassette in the proper reading frame. Generally, foreign DNA is inserted at one or more restriction sites of the vector DNA, and then is carried by the vector into a host cell along with the transmissible vector DNA. A segment or sequence of DNA having inserted or added DNA, such as an expression vector, can also be called a "DNA construct" or "gene construct." A common type of vector is a "plasmid", which generally is a self-contained molecule of double-stranded DNA, usually of bacterial origin, that can readily accept additional (foreign) DNA and which can readily introduced into a suitable host cell. A plasmid vector often contains coding DNA and promoter DNA and has one or more restriction sites suitable for inserting foreign DNA. Coding DNA is a DNA sequence that encodes a particular amino acid sequence for a particular protein or enzyme. Promoter DNA is a DNA sequence which initiates, regulates, or otherwise mediates or controls the expression of the coding DNA. Promoter DNA and coding DNA may be from the same gene or from different genes, and may be from the same or different organisms. A large number of vectors, including plasmid and fungal vectors, have been described for replication and/or expression in a variety of eukaryotic and prokaryotic hosts. Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids, pET plasmids (Novagen, Inc., Madison, WI), pRSET or pREP plasmids (Invitrogen, San Diego, CA), or pMAL plasmids (New England Biolabs, Beverly, MA), and many appropriate host cells, using methods disclosed or cited herein or otherwise known to those skilled in the relevant art. Recombinant cloning vectors will often include one or more replication systems for cloning or expression, one or more markers for selection in the host, e.g. antibiotic resistance, and one or more expression cassettes.

The term "host cell" means any cell of any organism that is selected, modified, transformed, grown, used or manipulated in any way, for the production of a substance by the cell, for example, the expression by the cell of a gene, a DNA or RNA sequence, a protein or an enzyme. Host cells can further be used for screening or other assays, as described herein.

A "polynucleotide" or "nucleotide sequence" is a series of nucleotide bases (also called "nucleotides") in a nucleic acid, such as DNA and RNA, and means any chain of two or more nucleotides. A nucleotide sequence typically carries genetic information, including the information used by cellular machinery to make proteins and enzymes. These terms include double or single stranded genomic and cDNA, RNA, any synthetic and genetically manipulated polynucleotide, and both sense and anti-sense polynucleotide. This includes single- and double- stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA hybrids, as well as "protein nucleic acids" (PNA) formed by conjugating bases to an amino acid backbone. This also includes nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluoro-uracil.

"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. The nucleic acids herein may be flanked by natural regulatory (expression control) sequences, or may be associated with heterologous sequences, including promoters, internal ribosome entry sites (IRES) and other ribosome binding site sequences, enhancers, response elements, suppressors, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like. The term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. The nucleic acids may also be modified by many means known in the art. Non-limiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, and carbamates) and with charged linkages (e.g., phosphorothioates, and phosphorodithioates). Polynucleotides may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, and poly-L-lysine), intercalators (e.g. , acridine, and psoralen), chelators (e.g., metals, radioactive metals, iron, and oxidative metals), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Modifications of the ribose- phosphate backbone may be done to facilitate the addition of labels, or to increase the stability and half-life of such molecules in physiological environments. Nucleic acid analogs can find use in the methods of the invention as well as mixtures of naturally occurring nucleic acids and analogs. Furthermore, the polynucleotides herein may also be modified with a label capable of providing a detectable signal, either directly or indirectly. Exemplary labels include radioisotopes, fluorescent molecules, and biotin.

The term "polypeptide" as used herein means a compound of two or more amino acids linked by a peptide bond. "Polypeptide" is used herein interchangeably with the term "protein."

The term "about" or "approximately" means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation. For example, "about" can mean within 1 or more than 1 standard deviations, per the practice in the art. Alternatively, "about" can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated, the term "about" meaning within an acceptable error range for the particular value should be assumed.

General Methods

Standard methods in molecular biology are described Sambrook, Fritsch and

Maniatis (1982 & 1989 2^nd Edition, 2001 3^rd Edition) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Sambrook and Russell (2001) Molecular Cloning, 3^rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Wu (1993) Recombinant DNA, Vol. 217, Academic Press, San Diego, CA). Standard methods also appear in Ausbel, et al. (2001) Current Protocols in Molecular Biology, Vols.1-4, John Wiley and Sons, Inc. New York, NY, which describes cloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast (Vol. 2), glycoconjugates and protein expression (Vol. 3), and bioinformatics (Vol. 4).

Methods for protein purification including immunoprecipitation, chromatography, electrophoresis, centrifugation, and crystallization are described (Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis, chemical modification, post-translational modification, production of fusion proteins, glycosylation of proteins are described (see, e.g., Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2, John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) Current Protocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY, NY, pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for Life Science Research, St. Louis, MO; pp. 45-89; Amersham Pharmacia Biotech (2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production, purification, and fragmentation of polyclonal and monoclonal antibodies are described (Coligan, et al. (2001) Current Protocols in Immunology, Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999) Using Antibodies, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Harlow and Lane, supra). Standard techniques for characterizing ligand/receptor interactions are available (see, e.g., Coligan, et al. (2001) Current Protocols in Immunology, Vol. 4, John Wiley, Inc., New York).

EXAMPLES

We performed Chromatin immunoprecipitation and sequencing (ChlP-seq) with a TOP2A and TOP2B antibody to investigate the genomic localization of TOP2B binding in glioma cell lines. TOP2B binding sites were enriched on the promoters of many genes including PDGFRA and MYC oncogenes on the proneural cell lines (TS543 and BT142) as opposed to cell lines SNB 19 and 0777A. Fig. 1A is graphic expression array data from human glioblastomas showing elevated expression of TOP2A and B in the proneural group (TCGA). The mesenchymal group have the lowest expression for TOP2A and B. The values for TOP2A% are shown as the left bar for each set; while the TOP2B% is shown as the right bar of each set. TOP2B binding on these oncogenes was associated with expression across these cell lines {See, Sonabend Adam M., et <a/.2014).

Figure IB, is a heatmap showing expression of genes identified as having TOP2B activity on promoters (defined as having binding on ChlP-seq) in the presence of TOP2 inhibitor ICRF193 or following treatment with DMSO (control). Of note, PDGFRA and MYC are down-regulated by TOP2 inhibition. In Figures 2A-B we show that TOP2A and TOP2B binding to PDGFRA gene in some glioma cell lines (Fig. 2A). Close up view of TOP2B binding to PDGFRA promoter and gene body determined by ChlP-seq (Fig. 2B left) and that this binding is associated with elevated expression across 4 human glioma cell lines (Fig. 2B right). Validation of these findings was done by ChlP-PCR (Fig. 2C, with IgG noted in the left bar of each set, and TOP2A shown in the right bar of each set for the left graph. For the right graph, IgG is noted in the left bar of each set and TOP2B data is shown in the right bar of each set). In Figure 3 we show TOP2A and TOP2B binding to MYC gene in some glioma cell lines (Fig. 3A). Close up view of TOP2B binding to MYC promoter and gene body determined by ChlP-seq (Fig. 3B left) and that this binding is associated with elevated expression across 4 human glioma cell lines (Fig. 3B right).

To explore whether there is a relationship between TOP2B and PDGFRA and MYC in human gliomas, we represented the expression of these genes from human gliomas on scatter plots, and found a correlation between TOP2B expression with the expression of PDGFRA and MYC. In Figure 4 we show gene expression data from human glioma patients (Gliovis, TCGA data) shows a correlation between TOP2B expression with PDGFRA and MYC.

To investigate the role of TOP2B in the transcription, we treated TS543 and BT142 with the TOP2 inhibitor ICRF-193 (10 μΜ) for 5 hr. To confirm the inhibition of TOP2 activity, we tested the enzymatic activity of TOP2A/B through a decatenation assay using nuclear extract from TS543 cells treated with DMSO or ICRF193.

In Figure 5 we show the results of a decatenation assay showing that a dose of 10 μΜ of TOP2 inhibitor ICRF193 significantly decreased the enzymatic activity of TOP2 on glioma cell line TS543. To confirm that genes with TOP2B binding do not suffer expression changes following TOP2 inhibition with ICRF193, we performed ChlP-PCR for two tumor suppressor genes Rbl and p53 and found no significant enrichment of

TOP2B on these genes, and showed that these genes do not change expression following ICRF193 treatment.

In Figure 6, we present the ChlP-PCR data showing no TOP2A/B binding to tumor-suppressor gene Rbl (Fig. 6A). TOP2 inhibition with ICRF193 had no significant effect on expression of oncogenes Rbl or p53 on multiple cell lines (Fig. 6B). Expression analysis of following 5 hr of TOP2 inhibition with ICRF193 showed a significant down- regulation of oncogenes PDGFRA and MYC on BT142 IDH^R132H (adj. p<7E-14), and similar results on TS543 IDH^wt PDGFRA^ glioma cell line.

In Figure 7 we show that TOP2 inhibition led to down-regulation of the mRNA

(top) and protein (bottom) of PDGFRA (left) and MYC (right). Transcript levels were determined by RNA-seq, and protein by Western Blot. MYC down-regulation was associated with down-regulation of its transcriptional targets following TOP2 inhibition.

In Figure 8, we present data showing that TOP2 inhibition led to impairment of

MYC activity demonstrated as down-regulation of MYC targets in glioma cells TS543 and BT142. Transcript levels were determined by RNA-seq. Analysis of expression was GSVA.We hypothesized that the expression levels of PDGFRA and MYC oncogenes could be used as a biomarker to predict the susceptibility to TOP2 inhibitors in gliomas.

First we validated that MST16 a TOP2A/B inhibitor currently used for lymphomas therapy has similar results than ICRF193 in BT142 as it downregulated these oncogenes.

In Figure 9, we show that treatment of human glioma cell lines with ICRF193 and the TOP2 inhibitor drug MST16 leads to significant down-regulation of PDGFRA and MYC determined by qRT-PCR.

We then tested the viability of two separate TOP2 inhibitor drugs on 8 human glioma cell lines. To investigate whether the effect on viability was related to an on-target effect on TOP2, we compared the susceptibility (expressed as the area unde the curve AUC) to these 2 distinct drugs that share their target, and found a high correlation between them. In Figure 10 we show that decrease in cell viability elicited by two separate TOP2 inhibitors is highly correlated, as cell viability following treatment with

TOP2 inhibitors is related to on-target effect.

Next, we performed a viability experiment and found variable susceptibility to TOP2 inhibitors (expressed as area under the curve, AUC) for MST-16 dose response curves. Susceptibility to the TOP2 inhibitor MST-16 (e.g. AUC) had a high correlation with the RNA-seq derived expression levels of oncogenes PDGFRA (Rho= -0.91, p=0.0014) and MYC (Rho= -0.7554, p=0.03) across 8 human glioma cell lines.

In Figure 11 we show the correlation between expression (RFPKM from RNA- seq data) of PDGFRA and MYC with susceptibility to TOP2 inhibitor MST-16

(determined by dose response area under the curve [AUC]). We then validated this finding using a collection of patient-derived glioma xenograft cell lines with wide range of PDGFRA expression. We observed that PDGFRA expression identified a cell line susceptible to MST16, suggesting that the expression of PDGFRA might be used as biomarker for MST16 susceptibility. In Figure 12, we show the validation of finding on an independent dataset of patient-derived glioma xenograft cell lines. Correlation between expression (RFPKM counts) of PDGFRA with susceptibility to TOP2 inhibitor MST-16 (determined by dose response area under the curve [AUC]).

Conclusions

These results show that TOP2A/B exerts epigenetic regulation of transcription on oncogenes PDGFRA and MYC in human gliomas. This is supported by its localization on promoters of oncogenes, and the associated decrease in expression of these genes following pharmacological inhibition. This work shows the therapeutic repurposing of existing drugs that inhibit TOP2 activity for treatment of human gliomas, and provide rationale for the use of MYC and PDGFRA expression as biomarkers for identifying susceptible tumors for this therapy. Targeting of TOP2 allows for the simultaneous therapeutic modulation of multiple oncogenes, exploiting the epigenetic mechanisms underlying chromatin structure in cancer. This information and data can also be applied to treating brain tumors and other cancers. Interestingly, TOP2 inhibition had no effect on the expression of p53 and Rbl tumor suppressors on most cell lines, which further reinforces the goal of this treatment modality to selectively target oncogenes in cancer. Methods

We performed ChlP-seq (Chromatin immunoprecipitation and sequencing) for TOP2A and B to investigate its genomic localization in proneural glioma genotypes TS543 PDGFRA^P and BT142 IDH1^R132H and two mesenchymal glioma cell lines SNB 19 and 0777A. RNA-seq and WB were used to evaluate gene expression. ChlP-seq and ChlP-qPCR was performed in the presence of etoposide to stabilize TOP2A/B genomic binding. TOP2 inhibition was performed with ICRF- 193 and confirmed by decatenation assays. Susceptibility to TOP2 inhibitor drug MST-16 was evaluated by area under the curve (AUC). To investigate therapeutic potential of TOP2 targeting, clinically available TOP2 inhibitors MST-16 and razoxane were compared. A T-test showing p<0.05 was considered significant, and DE-seq adjusted p <0.05 was defined as significant for RNA- seq data. The conditions of TOP2 inhibition were ΙΟμΜ ICRF-193 for an incubation time of 5 hours (RNA) and 48 hours (protein).

References

Tiwari et al, PNAS 2012; 109(16):E934-43.

Thakurela et al., Nature Comm 2013; 4:2478.

Kawagishi J, Kumabe T, Yoshimoto T, Yamamoto T. Genomics. 1995 Nov 20;30(2):224-32.

Sonabend Adam M., et al. Convection-enhanced delivery of etoposide is effective against murine proneural glioblastoma. Neuro Oncol. 16, 1210-1219 (2014).

Sonabend et al., Cancer Research 2014.

Methods

Human glioma cell lines were utilized for these studies. ChlP-qPCR and ChlP-seq were performed in the presence of etoposide to identify loci where TOP2A/B was enzymatically active. qRT-PCR and RNA-seq was used to evaluate gene expression +/- ICRF-193, a pharmacologic TOP2A/B inhibitor. Inhibition was confirmed by decatenation assays and decrease in protein concentration was investigated through Western blot.

These results demonstrate that inhibiting TOP2A/B expression using pharmacological agents such as ICRF-193 or MST-16 significantly decreases MYC and PDGFRA gene expression and protein level. Gene expression of MYC downstream targets also decreases 5 hours after TOP2 inhibition. Additionally, these results show that the efficacy of a TOP2 inhibitor is correlated with the gene expression levels of MYC and PDGFRA, as well as of TOP2A and TOP2B, in eight different glioblastoma cell lines. In particular these results indicate that PDGFRA and MYC may be clinically relevant biomarkers for identifying patients who will benefit from treatment with TOP2A/B inhibitors. In certain embodiments, the present disclosure provides for treatment for glioma patients with elevated level of MYC and/or PDGFRA expression with TOP2A/B inhibitors. Additionally, this treatment could be combined with other therapeutics for more effective glioma therapy. In additional embodiments, treatment for other types of cancer or disease characterized by MYC and/or PDGFRA overexpression is provided. Finally, the present disclosure also provides for diagnostic assays to identify patients who would benefit from treatment with a topoisomerase inhibitor.

This technology identifies topoisomerase II inhibition as a potential treatment strategy for a subset of gliomas with MYC and PDGFRA overexpression. Topoisomerases are enzymes that have been shown to globally regulate gene expression in neurons and stem cells. This technology demonstrates that topoisomerase II enzymatic activity is enriched at the promoter regions of MYC and PDGFRA genes, both of which are important oncogenes that drive glioma growth. Treating glioblastoma cell lines with a topoisomerase II inhibitor has been shown to decrease MYC and PDGFRA gene expression and reduce the viability of glioma cell lines. The efficacy of a topoisomerase II inhibitor in killing glioblastoma cells is directly correlated with MYC and PDGFRA expression levels. These results demonstrate the potential for topoisomerase II inhibitor- based therapies for treatment of gliomas and other diseases associated with MYC and/or PDGFRA overexpression.

Such assays would include a method for for inhibiting tumor growth in a patient in need thereof, comprising determining expression levels in the tumor of one or any combination of PDGFRA expression and MYC expression, wherein overexpression of PDGFRA, MYC, or a combination thereof, above a baseline or reference level indicates a tumor likely to be sensitive to topoisomerase inhibitor treatment; and in such instances administering an effective amount of a topoisomerase inhibitor to the patient. Such a reference level can be attained from correlated expression data as shown in Fig. 11 and Fig. 12.

All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g. Genbank sequences or GenelD entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. § 1.57(b)(1), to relate to each and every individual publication, database entry (e.g. Genbank sequences or GenelD entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. § 1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims. The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims.

Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The invention is defined by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. The specific embodiments described herein, including the following examples, are offered by way of example only, and do not by their details limit the scope of the invention.

Claims

WHAT IS CLAIMED IS:

I . A method for inhibiting tumor growth in a patient in need thereof, comprising administering an effective amount of a topoisomerase inhibitor to the patient.

2 The method of claim 2, wherein the topoisomerase inhibitor comprises a topoisomerase II (TOP2) inhibitor.

3. The method of claim 2, wherein the TOP2 inhibitor comprises TOP2A/B.

4. The method of claim 3, wherein the TOP2A/B inhibitor comprises ICFR-193, MST-6, ICFR-154, razoxane or any combination thereof.

5. The method of claim 1, wherein the tumor comprises a brain tumor.

6. The method of claim 5, wherein the brain tumor is a glioma.

7. The method of clalim 1, wherein prior to administering, the tumor is tested for PDGFRA expression, MYC expression, or a combination thereof, wherein

overexpression of PDGFRA, MYC, or acombination thereof, above a baseline or reference level indicates a tumor likely to be sensitive to TOP2 inhibitor treatment.

8. A method for inhibiting PDGFRA and MYC oncogenes in a glioma tumor, comprising contacting the glioma tumor with an effective amount of a topoisomerase inhibitor.

9. The method of claim 8, wherein the topoisomerase inhibitor comprises a TOP2 inhibitor.

10. The method of claim 9, wherein the TOP2 inhibitor comprises a TOP2A/B inhibitor.

I I. The method of claim 10, wherein the TOP2A/B inhibitor comprises ICFR-193, MST-16,ICFR-154, or razoxane, or any combination thereof.

12. The method of claim 8, wherein prior to administering, the tumor is tested for PDGFRA expression, MYC expression, or a combination thereof, wherein

overexpression of PDGFRA, MYC, or a combination thereof, above a baseline or reference level indicates a tumor likely to be sensitive to TOP2 inhibitor treatment.

13. The method of claim 8, further comprising administering at least one additional chemotherapeutic agent and/or radiation.

14. The method of claim 1, further comprising administering at least one additional chemotherapeutic agent and/or radiation.

15. A method for for inhibiting tumor growth in a patient in need thereof, comprising determining expression levels in the tumor of one or any combination of PDGFRA expression and MYC expression, wherein overexpression of PDGFRA, and/or MYC above a baseline or reference level indicates a tumor likely to be sensitive to

topoisomerase inhibitor treatment; and in such instances administering an effective amount of a topoisomerase inhibitor to the patient.

16. The method of claim 15, wherein the topoisomerase inhibitor comprises a topoisomerase II (TOP2) inhibitor.

17. The method of claim 16, wherein the TOP2 inhibitor comprises TOP2A/B.

18. The method of claim 17, wherein the TOP2A/B inhibitor comprises ICFR-193, MST-6, ICFR-154, razoxane, or any combination thereof.

19. The method of claim 15, wherein the tumor comprises a brain tumor.

20. The method of claim 19, wherein the brain tumor is a glioma.

21. The method of claim 15, further comprising administering at least one additional chemotherapeutic agent and/or radiation.