WO2016172332A1 - A novel compound for the treatment of ewing sarcoma and high-throughput assays for identifying small molecules that modulate aberrant chromatin accessibility - Google Patents

Abstract

The present invention provides a compound useful for the treatment of cancer, and in particular, treatment of Ewing sarcoma. The present invention further provides an assay for identifying a compound that modulates chromatin activity of EWS-FLI1.

Description

A NOVEL COMPOUND FOR THE TREATMENT OF EWING SARCOMA AND HIGH-THROUGHPUT ASSAYS FOR IDENTIFYING SMALL MOLECULES THAT

MODULATE ABERRANT CHROMATIN ACCESSIBILITY

Statement of Government Support

This invention was made with government support under grant numbers R01CA166447, RC1GM090732 and R01GM100919 from the National Institutes of Health. The government has certain rights to this invention.

Field of the Invention

The present invention relates to a compound that modulates aberrant chromatin accessibility and can be used to treat various disorders, such as cancer. The present invention also relates to additional methods of using the compound and assays for identifying compounds, in particular, small molecules, that modulate aberrant chromatin accessibility.

Background of the Invention

Ewing sarcoma is a highly malignant pediatric bone and soft tissue tumor. The majority of Ewing sarcoma tumors harbors the chromosomal translocation t(l l;22)(q24;ql2), which brings together EWSRl with the ETS transcription factor family member FLU generating an EWS-FLI1 chimeric transcription factor (Delattre et al. 1992). A major challenge facing the biological targeting of aberrant transcriptional regulators such as EWS- FLI1 is that transcription factors lacking enzymatic activity or binding pockets with targetable molecular features have typically been considered "undruggable," and a reductionist approach based on identification of their molecular targets has largely failed.

Summary of the Invention

Embodiments of present invention are directed to a compound having the following structure:

and pharmaceutically acceptable salts thereof.

Embodiments of the present invention provide the compounds described herein in pharmaceutical compositions including a pharmaceutically acceptable carrier and formulated for various delivery modes.

Embodiments of the present invention provide methods of treating cancer including administering an effective amount of the compounds described herein.

Embodiments of the present invention provide methods of reducing chromatin accessibility, inhibiting proliferation of a Ewing sarcoma cell, inhibiting transformation into a Ewing sarcoma cell and/or decreasing Ewing sarcoma cell viability.

Embodiments of the present invention provide an in vitro method for identifying a compound that modulates aberrant chromatin accessibility at a specific genomic locus based on the activity of an oncogene for the manufacture of a diagnostic or therapeutic agent for cancer.

Embodiments of the present invention further provide use of the compounds described herein for manufacture of a pharmaceutical or medicament. The pharmaceutical or medicament can be used to treat cancer as described herein.

Brief Description of the Drawings

Figure 1. Comparison of FAIRE methodologies, (a) Heatmap representation of normalized Formaldehyde- Assisted Isolation of Regulatory Elements (FAIRE) enrichment (± 3 kb from TSS) using standard (left) or column (right) FAIRE in HUVEC. (b) Normalized FAIRE signal from both methods ± 3 kb from TSS. (c) Normalized FAIRE signal from both methods ± 3 kb around HUVEC CTCF sites (ENCODE), (d) Fraction of top 10,000 HT- FAIRE enriched sites overlapping standard FAIRE sites from HUVEC and 6 other cell types (ENCODE), (e) Hierarchical clustering analysis of 500 bp intervals demonstrating differential FAIRE signal across 7 cell types as well as HUVEC HT-FAIRE. Platform- specific (Clusters 1 and 3) and cell-type-specific (Cluster 2) clusters were identified, (f) Fractional overlap annotation of Clusters 1-3 with histone modifications and transcription factors (ENCODE), (g) Fractional overlap annotation of Clusters 1-3 with repetitive element classes.

Figure 2. Chromatin signature-based screen identified UNC0621 as an inhibitor of EWS-FLI-dependent chromatin accessibility, (a) Flow diagram comparing column- based and standard FAIRE methods, (b-c) The chromatin-focused library consists of two 384-well compound plates. Plate 1 (b) or Plate 2 (c) log₂ ratio of the relative chromatin inhibition values was plotted against the rank order of compounds from greatest relative decrease (top, left side of X-axis) to the greatest relative increase (top, right side of X-axis) in FAIRE signal following compound treatment. The dashed lines indicate the significance cutoff of relative chromatin inhibition values greater or less than two standard deviations from the average FAIRE signal for vehicle-treated controls. Thirty compounds that show the greatest decrease in FAIRE signal for each plate are magnified (bottom graph). The bar representing UNC0621 is highlighted in black with an asterisk (*). (d) Chemical structure of UNC0621. (e) Standard FAIRE-qPCR at 11 genomic loci known to be enriched by FAIRE in EWS894 cells following 16-hr treatment with 10 μΜ UNC0621. "Target" sites were dependent on the binding and presence of EWS-FLI1 for a positive FAIRE signal, whereas "Control" sites were not EWS-FLI1 -dependent. Error bars represent the standard deviation of three biological replicates, (f) HT-FAIRE-qPCR at the same genomic regions used for the original screen was conducted following treatment of EWS894 cells for 16 hours with 3-fold dilutions of UNC0621 or a control compound in concentrations ranging from 1.11 to 0.01 μΜ. Error bars represent the standard deviation of 4 replicates for UNC0621 and the standard error of 2 replicates for the control compound.

Figure 3. UNC0621 affects proliferation, viability and transformation of Ewing Sarcoma cells. Dose-dependent effect of UNC0621 on the viability of (a) Ewing Sarcoma cell lines, (b) renal cell carcinoma cell lines, and (c) human primary cells. Cell viability was assayed using WST-1 reagent (Roche) and spectrophotometry. Background absorbance (620 nm) was subtracted from assay absorbance (450 nm) values. Relative cell viability is expressed as UNC0621^Abs450"620/DMSO^Abs450"620. Error bars represent standard deviation of three biological replicates, (d) Cells were cultured in the presence of 10 μΜ UNC0621 or 0.1 % DMSO for six days, and viability was assessed daily using WST-1 reagent and spectrophotometry. Values are background corrected as above. Error bars represent standard deviation of three biological replicates, (e) Cells were grown in the presence of 10 μΜ UNC0621 or 0.1% DMSO for 3 days. Equal numbers of viable cells were then re-plated in the growth medium in the absence of compound and counted each day for 6 days. Error bars represent standard deviation of triplicate cell counts, (f) Effect of U C0621 on anchorage- independent growth of Ewing Sarcoma cells. EWS894 cells were plated in agar-containing growth media containing UNC0621 or DMSO and incubated for 15 days. Fresh media containing UNC0621 was overlayed and changed every 5 days. Colony formation was assessed on day 15 by MTT assay, (g) EWS894 soft agar colony counts. Error bars represent standard deviation of three biological replicates.

Detailed Description

The present invention is further described below in greater detail. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following specification is intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

All patent and patent application references referred to in this patent application are hereby incorporated by reference in their entirety as if set forth fully herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprise," "comprises" and "comprising" as used herein, specify the presence of the stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof.

As used herein, the transitional phrase "consisting essentially of means that the scope of a claim is to be interpreted to encompass the specified materials or steps recited in the claim and those that do not materially affect the basic and novel characteristic(s) of the claimed invention. Thus, the term "consisting essentially of when used in a claim of this invention is not intended to be interpreted to be equivalent to "comprising."

As used herein, "a," "an" or "the" can mean one or more than one. Also as used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or"). The term "about," as used herein when referring to a measurable value such as an amount of dose (e.g., an amount of a compound) and the like, is meant to encompass variations of t 20%, ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of the specified amount.

An "acid" is a compound that can act as a proton donor or electron pair acceptor, and thus can react with a base. The strength of an acid corresponds to its ability or tendency to lose a proton. A "strong acid" is one that completely dissociates in water. Examples of strong acids include, but are not limited to, hydrochloric acid (HC1), hydroiodic acid (HI), hydrobromic acid (HBr), perchloric acid (HCIO₄), nitric acid (HN0₃), sulfuric acid (H₂S0₄), etc. A "weak" or "mild" acid, by contrast, only partially dissociates, with both the acid and the conjugate base in solution at equilibrium. Examples of mild acids include, but are not limited to, carboxylic acids such as acetic acid, citric acid, formic acid, gluconic acid, lactic acid, oxalic acid, tartaric acid, ethylenediaminetetraacetic acid (EDTA), etc.

"Aqueous" refers to a solution in which water is the dissolving medium, or solvent. An "aqueous base" is a base in water. An "aqueous acid" is an acid in water.

A "base" is a compound that can accept a proton (hydrogen ion) or donate an electron pair. A base may be organic (e.g., DBU, cesium carbonate, etc.) or inorganic. A "strong base" as used herein is a compound that is capable of deprotonating very weak acids. Examples of strong bases include, but are not limited to, hydroxides, alkoxides, and ammonia.

As used herein, "nucleic acid" and "nucleotide sequence" and "polynucleotide: encompass both RNA and DNA, including cDNA, genomic DNA, mRNA, synthetic (e.g., chemically synthesized) DNA and chimeras of RNA and DNA. The term polynucleotide or nucleotide sequence refers to a chain of nucleotides without regard to length of the chain. The nucleic acid can be double-stranded or single-stranded. Where single-stranded, the nucleic acid can be a sense strand or an antisense strand. The nucleic acid can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases. The present invention further contemplates a nucleic acid that is the complement (which can be either a full complement or a partial complement) of a nucleic acid or nucleotide sequence of those described in relation to this invention.

The term "oligonucleotide" refers to a nucleic acid sequence of at least about five nucleotides to about 100 nucleotides, for example, about 12 to 18, about ί5 to 30 nucleotides, or about 20 to 25 nucleotides, which can be used, for example, as a primer in a PCR amplification and/or as a probe in a hybridization assay or in a microarray. Oligonucleotides can be natural or synthetic, e.g., DNA, RNA, modified backbones, etc.

The present invention further contemplates fragments or oligonucleotides of the nucleic acids discussed in relation to this invention, which can be used, for example as primers or probes and as antisense sequences. Thus, in some embodiments, a fragment or oligonucleotide is a nucleotide sequence that comprises, consists essentially of and/or consists of at least, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110. 120, 125, 135, 150, 160, 170, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 or 500 contiguous nucleotides of a specified nucleic acid. Such fragments or oligonucleotides can be detectably labeled or modified, for example, to include and/or incorporate a restriction enzyme cleavage site when employed as a primer in an amplification (e.g., PCR) assay.

The present invention may also relate to isolated polypeptides, peptides, proteins and/or fragments that are substantially equivalent to those described in relation to this invention. As used herein, "substantially equivalent" can refer both to nucleic acid and amino acid sequences, for example a mutant sequence, that varies from a reference sequence by one or more substitutions (e.g., substitution with conservative amino acids as are well known in the art), deletions and/or additions, the net effect of which does not result in an undesirable adverse functional dissimilarity between reference and subject sequences. In some embodiments, this invention can include substantially equivalent sequences that have an adverse functional dissimilarity. For purposes of the present invention, sequences having equivalent biological activity and equivalent expression characteristics are considered substantially equivalent.

The invention further provides homologues, as well as methods of obtaining homologues, of the polypeptides and/or fragments of those described in relation to this invention from other species. As used herein, an amino acid sequence or protein is defined as a homologue of a polypeptide or fragment of those described in relation to the present invention if it shares significant homology to one of the polypeptides and/or fragments of the present invention. Significant homology means at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% and/or 100% homology with another amino acid sequence. Specifically, by using the nucleic acids that encode the proteins and fragments described in relation to this invention, as a probe or primer, and techniques such as PCR amplification and colony/plaque hybridization, one skilled in the art can identify homologues of the polypeptides and/or fragments described in relation to this invention in other organisms.

"Substantial homology or similarity" means that a nucleic acid or fragment thereof is "substantially homologous" (or "substantially similar") to another if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), using, e.g., the BLASTN alignment program, there is nucleotide sequence identity in at least about 60% of the nucleotide bases, usually in at least about 70%, more usually in at least about 80%, in at least about 90%, or in at least about 95-98% of the nucleotide bases. To determine homology between two different nucleic acids, the percent homology can be determined using the BLASTN program "BLAST 2 sequences." This program is available for public use from the National Center for Biotechnology Information (NCBI) over the internet (Altschul et al., 1997). The parameters to be used are whatever combination of the following yields the highest calculated percent homology (as calculated below) with the default parameters shown in parentheses: Program—blastn Matrix~0 BLOSUM62 Reward for a match-0 or 1 (1) Penalty for a mismatch-0, -1, -2 or -3 (-2) Open gap penalty— 0, 1 , 2, 3, 4 or 5 (5) Extension gap penalty— 0 or 1 (1) Gap x_dropoff— 0 or 50 (50) Expect— 10.

The terms "substantial homology" or "substantial identity", when referring to polypeptides, indicate that the polypeptide or protein in question exhibits at least about 50% identity using the BLASTP program with an entire naturally-occurring protein or a portion thereof, usually at least about 70% identity over the common lengths, more usually at least about 80% identity, at least about 90% identity, or at least about 95% identity.

Homology, for polypeptides, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measures of homology assigned to various substitutions, deletions and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

As used herein, "portion" or "fragment" are used interchangeably and refers to less than the whole of the structure that substantially retains at least one biological activity normally associated with that molecule, protein or polypeptide. In particular embodiments, the "fragment" or "portion" substantially retains all of the activities possessed by the unmodified protein. By "substantially retains" biological activity, it is meant that the protein retains at least about 50%, 60%, 75%, 85%, 90%, 95%, 97%, 98%, 99%, or more, of the biological activity of the native protein (and can even have a higher level of activity than the native protein).

The term "locus" refers to a fixed position in a genome corresponding to a gene. A locus may have an associated "locus control region" which refers to a segment of DNA that controls the chromatin structure and thus the potential for replication and transcription of an entire gene cluster.

As used herein, "nuclease" refers to any of several enzymes that hydrolize nucleic acids. Nucleases may be non-specific, specific for types of nucleic acid such as DNA or R A, and/or specific for single or double stranded forms of nucleic acids. Nucleases include various overlapping categories of enzymes, for example, deoxynucleases, which specifically hydrolize DNA, and endonucleases which are nucleases that cleave nucleic acids at interior bonds and so produce fragments of various sizes. In particular embodiments of the present invention, the nuclease is specific for RNA.

A "peptide" is a linear chain of amino acids covalently linked together, typically through an amide linkage, and contains from 1 or 2 to 10 or 20 or more amino acids, and is also optionally substituted and/or branched.

As used herein, "modulate" or "modulation" refers to enhancement (e.g., an increase) or inhibition (e.g., a reduction) in the activity of interest. Those skilled in the art will appreciate that inhibition or reduction does not require complete cessation of the activity of interest.

"Effective amount" as used herein refers to an amount of a compound, composition or formulation of the invention that is sufficient to produce a desired effect, which can be a therapeutic and/or beneficial effect. The effective amount will vary with the age, general condition of the subject, the severity of the condition being treated, the particular agent administered, the duration of the treatment, the nature of any concurrent treatment, the pharmaceutically acceptable carrier used, and like factors within the knowledge and expertise of those skilled in the art. As appropriate, an "effective amount" in any individual case can be determined by one of ordinary skill in the art by reference to the pertinent texts and literature and/or by using routine experimentation.

By the term "treat," "treating" or "treatment of (and grammatical variations thereof) it is meant that the severity of the subject's condition is reduced, at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved and/or there is a delay in the progression of the disease or disorder. Treat does not necessarily indicate a cure.

A "treatment effective" amount as used herein is an amount that is sufficient to treat (as defined herein) the subject. Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject.

"Therapeutic" refers to an agent, drug, compound, composition or the like that imparts a desired biological, physiological and/or pharmacological effect, which need not be complete or curative, as long as some benefit is provided.

The term "prevent," "preventing" or "prevention of (and grammatical variations thereof) refer to prevention and/or delay of the onset and/or progression of a disease, disorder and/or a clinical symptom(s) in a subject and/or a reduction in the severity of the onset and/or progression of the disease, disorder and/or clinical symptom(s) relative to what would occur in the absence of the methods of the invention. In representative embodiments, the term "prevent," "preventing," or "prevention of (and grammatical variations thereof) refer to prevention and/or delay of the onset and/or progression of a metabolic disease in the subject, with or without other signs of clinical disease. The prevention can be complete, e.g., the total absence of the disease, disorder and/or clinical symptom(s). The prevention can also be partial, such that the occurrence of the disease, disorder and/or clinical symptom(s) in the subject and/or the severity of onset and/or the progression is less than what would occur in the absence of the present invention.

A "prevention effective" amount as used herein is an amount that is sufficient to prevent (as defined herein) the disease, disorder and/or clinical symptom in the subject. Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some benefit is provided to the subject.

"Diagnostic" as used herein refers to the use of information (e.g., genetic information or data from other molecular tests on biological samples, signs and symptoms, physical exam findings, cognitive performance results, etc.) to anticipate the most likely outcomes, timeframes, and/or response to a particular treatment for a given disease, disorder, or condition, based on comparisons with a plurality of individuals sharing common nucleotide sequences, symptoms, signs, family histories, or other data relevant to consideration of a subject's health status. Accordingly, as "diagnostic agent" refers to a molecule, agent, drug, compound, composition or the like that allows one to obtain and/or use the information referenced above. "Transformation" as used herein refers to conversion of a normal cell to a tumor cell. The transformation may involve functional and/or genetic changes associated with the conversion of the normal cell into a tumor cell. The term "tumor," as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

"Proliferation" as used herein refers to a level of cell division, cell death, or both for the given cell type. In many cases, tumor cells proliferate more rapidly than normal cells.

As used herein, "viability" refers to the health of a cell or population of cells. Parameters that can be used to define viability include redox potential, cell membrane integrity and metabolism, i.e., evaluating properties and/or function.

Embodiments of the present invention provide a compound having the following structure:

Additionally, the present invention provides pharmaceutically acceptable salts of the compound. Pharmaceutically acceptable salts of the compound include a salt form of the compound of the present invention that permits their use or formulation as pharmaceuticals and which retains the biological effectiveness of the free acids and bases of the specified compound and that is not biologically or otherwise undesirable. Examples of such salts are described in Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wermuth, C.G. and Stahl, P.H. (eds.), Wiley- Verlag Helvetica Acta, Zurich, 2002 [ISBN 3-906390-26- 8]. Examples of such salts include alkali metal salts and addition salts of free acids and bases. Examples of pharmaceutically acceptable salts, without limitation, include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogenphosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-l,4-dioates, hexyne-l,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, γ- hydroxybutyrates, glycollates, tartrates, methanesulfonates, ethane sulfonates, propanesulfonates, toluenesulfonates, naphthalene- 1 -sulfonates, naphthalene-2-sulfonates, and mandelates.

Embodiments of the present invention also provide pharmaceutical compositions that include the compound described herein and a pharmaceutically acceptable carrrier. The pharmaceutical composition may further include additives such as binders, excipients, disintegrating agents, lubricants, glidants, sweeteners and/or flavoring agents. For example, pharmaceutical compositions of the present invention may include a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring. The particular choice of carrier and additives as well as formulation will depend upon the particular route of administration for which the composition is intended.

The compositions of the present invention may be suitable for and formulated for parenteral, oral, inhalation spray, topical (i.e., both skin and mucosal surfaces, including airway surfaces), rectal, nasal, buccal (e.g., sub-lingual), vaginal or implanted reservoir administration, etc. where the most suitable route in any given case will depend on the nature and severity of the condition being treated in combination with the drug profile of the compound described herein as would be understood by one of ordinary skill in the art.

The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraperitoneal, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

Compositions for injection will include the active ingredient together with suitable carriers including propylene glycol-alcohol-water, isotonic water, sterile water for injection (USP), emulPhor™-alcohol-water, cremophor-EL™, polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil, with other additives for aiding solubility or preservation may also be included, or other suitable carriers known to those skilled in the art. Accordingly, these carriers may be used alone or in combination with other conventional solubilizing agents such as ethanol, propylene glycol, or other agents known to those skilled in the art.

Compositions for oral administration may be, for example, solid preparations such as tablets, sugar-coated tablets, hard capsules, soft capsules, granules, powders, gelatins, and the like, with suitable carriers and additives being starches, sugars, binders, diluents, granulating agents, lubricants, disintegrating agents and the like. Because of their ease of use and higher patient compliance, tablets and capsules represent the most advantageous oral dosage forms for many medical conditions.

Similarly, compositions for liquid preparations include solutions, emulsions, dispersions, suspensions, syrups, elixirs, and the like with suitable carriers and additives being water, alcohols, oils, glycols, preservatives, flavoring agents, coloring agents, suspending agents, and the like.

Where the compounds described herein are to be applied in the form of solutions or injections, the compounds may be used by dissolving or suspending in any conventional diluent. The diluents may include, for example, physiological saline, Ringer's solution, an aqueous glucose solution, an aqueous dextrose solution, an alcohol, a fatty acid ester, glycerol, a glycol, an oil derived from plant or animal sources, a paraffin and the like. These preparations may be prepared according to any conventional method known to those skilled in the art.

Compositions for nasal administration may be formulated as aerosols, drops, powders and gels. Aerosol formulations typically comprise a solution or fine suspension of the active ingredient in a physiologically acceptable aqueous or non-aqueous solvent. Such formulations are typically presented in single or multidose quantities in a sterile form in a sealed container. The sealed container can be a cartridge or refill for use with an atomizing device. Alternatively, the sealed container may be a unitary dispensing device such as a single use nasal inhaler, pump atomizer or an aerosol dispenser fitted with a metering valve set to deliver a therapeutically effective amount, which is intended for disposal once the contents have been completely used. When the dosage form comprises an aerosol dispenser, it will contain a propellant such as a compressed gas, air as an example, or an organic propellant including a fluorochlorohydrocarbon or fluorohydrocarbon.

Compositions suitable for buccal or sublingual administration include tablets, lozenges, gelatins, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth or gelatin and glycerin.

In particular embodiments, the present invention provides a pharmaceutical formulation including the compound described herein wherein the pharmaceutical formulation is a parenteral formulation. In some embodiments, the parenteral formulation is an intravenous formulation. In some embodiments the parenteral formulation is an intraperitoneal formulation. In other embodiments, the present invention provides a pharmaceutical formulation including the compound described herein wherein the pharmaceutical formulation is an oral formulation.

Embodiments of the present invention also provide a method of treating cancer including administering an effective amount of the compound described herein to a subject in need thereof.

"Cancer" refers to an abnormal growth of cells which tends to proliferate in an uncontrolled way and, in some cases, to metastasize (i.e., spread). Specific cancer types include without limitation the following: Cardiac: sarcoma (e.g., such as angiosarcoma, fibrosarcoma, rhabdomyosarcoma, liposarcoma and the like), myxoma, rhabdomyoma, fibroma, lipoma and teratoma. Lung: bronchogenic carcinoma (e.g., such as squamous cell, undifferentiated small cell, undifferentiated large cell, adenocarcinoma and the like), alveolar (e.g., such as bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma, chondromatous hamartoma, mesothelioma. Gastrointestinal: esophagus (e.g., such as squamous cell carcinoma, adenocarcinoma, leiomyosarcoma, lymphoma and the like), stomach (e.g., such as carcinoma, lymphoma, leiomyosarcoma and the like), pancreas (e.g., such as ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumors, vipoma and the like), small bowel (e.g., such as adenocarcinoma, lymphoma, carcinoid tumors, Karposi sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, fibroma, and the like), large bowel (e.g., such as adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, leiomyoma and the like). Genitourinary tract: kidney (e.g., such as adenocarcinoma, Wilms tumor nephroblastoma, lymphoma, leukemia, and the like), bladder and urethra (e.g., such as squamous cell carcinoma, transitional cell carcinoma, adenocarcinoma and the like), prostate (e.g., such as adenocarcinoma, sarcoma), testis (e.g., such as seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma, fibroadenoma, adenomatoid tumors, lipoma and the like). Liver: hepatoma (e.g., hepatocellular carcinoma and the like), cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma, hemangioma. Bone: osteogenic sarcoma (e.g., such as osteosarcoma and the like), fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing sarcoma, malignant lymphoma (e.g., such as reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor chordoma, osteochronfroma (e.g., such as osteocartilaginous exostoses), benign chondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma and giant cell tumors. Nervous system: skull (e.g., such as osteoma, hemangioma, granuloma, xanthoma, osteitis deformans and the like), meninges (e.g., such as meningioma, meningiosarcoma, gliomatosis and the like), brain (e.g., such as astrocytoma, medulloblastoma, glioma, ependymoma, germinoma [pinealoma], glioblastoma multiform, oligodendroglioma, schwannoma, retinoblastoma, congenital tumors and the like), spinal cord (e.g., such as neurofibroma, meningioma, glioma, sarcoma and the like). Gynecological: uterus (e.g., such as endometrial carcinoma and the like), cervix (e.g., such as cervical carcinoma, pre-tumor cervical dysplasia and the like), ovaries (e.g., such as ovarian carcinoma [serous cystadenocarcinoma, mucinous cystadeno carcinoma, unclassified carcinoma], granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma, malignant teratoma, and the like), vulva (e.g., such as squamous cell carcinoma, intraepithelial carcinoma, adenocarcinoma, fibrosarcoma, melanoma and the like), vagina (e.g., such as clear cell carcinoma, squamous cell carcinoma, botryoid sarcoma (embryonal rhabdomyosarcoma], fallopian tubes (carcinoma) and the like). Hematologic: blood (e.g., such as myeloid leukemia [acute and chronic], acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative diseases, multiple myeloma, myelodysplastic syndrome and the like), Hodgkin's disease, non-Hodgkin's lymphoma. Sskin: malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Karposi's sarcoma, moles dysplastic nevi, lipoma, angioma, dermatofibroma, keloids, psoriasis and the like. Adrenal glands: neuroblastoma.

In particular embodiments, the cancer is one of a Ewing family of tumors (EFTs). The Ewing family of tumors is a group of cancers that includes Ewing sarcoma whether of bone (ETB or Ewing sarcoma of bone) or of extraosseous location Ewing tumors (EOE tumors). These tumors may also be referred to as primitive neuroectodermal tumors (PNET or peripheral neuroepithelioma), and Askin tumors (PNET of the chest wall). These tumors are thought to originate from the same type of stem cell. In specific embodiments, the cancer is a Ewing sarcoma.

As used herein, the terms "subject" and "patient" are used interchangeably and refer to those to be treated according to the present invention including any subject in whom prevention and/or treatment of cancer is needed or desired, as well as any subject prone to such a disorder. In some embodiments, the subject is a human; however, a subject of this invention can include an animal subject, particularly mammalian subjects such as canines, felines, bovines, caprines, equines, ovines, porcines, rodents (e.g. rats and mice), lagomorphs, primates (including non-human primates), etc., including domesticated animals, companion animals and wild animals for veterinary medicine or treatment or pharmaceutical drug development purposes. The subjects relevant to this invention may be male or female and may be any species and of any race or ethnicity, including, but not limited to, Caucasian, African-American, African, Asian, Hispanic, Indian, etc., and combined backgrounds. The subjects may be of any age, including newborn, neonate, infant, child, adolescent, adult, and geriatric. In some embodiments, the subject is one who has been diagnosed with or is suspected of having Ewing sarcoma. In some embodiments, the subject is one in whom other cancer treatment modalities have failed.

Embodiments of the present invention further provide a method of reducing chromatin accessibility, inhibiting proliferation of a Ewing sarcoma cell or cell from a Ewing sarcoma cell line, inhibiting transformation into a Ewing sarcoma cell or cell from a Ewing sarcoma cell line and/or decreasing the viability of a Ewing sarcoma cell or cell from a Ewing sarcoma cell line.

"Chromatin" refers to a structure comprised of DNA and proteins into which a eukaryotic genome is tightly packed. In general, the structural unit of chromatin or nucleosome, is composed of at least five types of histones (designated HI, H2A, H2B, H3, and H4) and approximately 1.8 turns of DNA wound around a core particle of the histone proteins. Positioning of nucleosomes throughout a genome can have a regulatory function by modifying the availability of binding sites, i.e., "chromatin accessibility," to transcription factors and overall transcription mechanisms. Modulation of the chromatin structure to increase the accessibility of DNA for protein interaction has been implicated in genetic disorders, including cancer.

According to some embodiments of the present invention, the compound described herein reduces the chromatin accessibility mediated by the EWS-FLI1 chimeric transcription factor by reversing aberrant chromatin activity associated with EWS-FLI1 at signature regions as discussed below. Embodiments of the present invention also inhibit proliferation of Ewing sarcoma cells or cells from a Ewing sarcoma cell line, for example, by inhibiting, slowing the progression of or halting cell growth. Embodiments of the present invention further inhibit the transformation of a Ewing sarcoma cell or cell from a Ewing sarcoma cell line by, for example, inhibiting, slowing the progression of or halting the conversion of a normal cell to a Ewing sarcoma cell. Embodiments of the present invention also decrease the viability of Ewing sarcoma cell or cell from a Ewing sarcoma cell line by, for example, inhibiting the proliferation of these cells, inhibiting the transformation of these cells, decreasing the metabolic capacity of these cells, decreasing the function of these cells required for their survival and/or negatively impacting a chemical or physical property of these cells.

These methods of reducing chromatin accessibility, inhibiting proliferation of a Ewing sarcoma cell, inhibiting transformation of a Ewing sarcoma cell and/or decreasing Ewing sarcoma cell viability include contacting a target cell with the compound described herein. In embodiments of the present invention, the target cell is a cancer cell. In further embodiments, the cell is derived from a Ewing sarcoma cell line or is a Ewing sarcoma cell.

Embodiments of the present invention further provide an adapted approach to isolating active regulatory elements from chromatin. In particular, Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) as described by Giresi et al. (2007) provides a procedure for the isolation of nucleosome-depleted DNA from human chromatin in which chromatin is crosslinked with formaldehyde in vitro, sheared by sonication, and phenol- chloroform extracted. The DNA recovered in the aqueous phase is fluorescently labeled and hybridized to a DNA microarray, sequenced or otherwise quantitated by methods including but not limited to quantitative polymerase chain reaction. Embodiments of the present invention relate to an adapted and automated FAIRE process to provide a high-throughput chromatin screen examining variation in FAIRE signal in response to cellular perturbations, including small molecule treatments.

Accordingly, embodiments of the present invention provide an in vitro method for identifying a compound that modulates aberrant chromatin accessibility at at least one specific genomic locus for the manufacture of a diagnostic or therapeutic agent for cancer or other medical condition associated with variation in chromatin accessibilty, the method including (a) contacting a sample including the at least one specific genomic locus with a compound of interest, wherein in some embodiments, the sample includes a Ewing sarcoma cell, a cell from a Ewing sarcoma cell line, or a combination thereof; (b) contacting the sample with formaldehyde; (c) obtaining cells from (b) and suspending the cells in a buffer (for example, an aqueous buffer); (d) sonicating the buffer; (e) adding a nuclease to the buffer from (d); (f) subjecting the buffer from (e) to a solid-phase support system; (g) eluting a nucleic acid from the solid-phase support system to provide nucleosome depleted regions of chromatin; and (h) comparing the enrichment at the at least one specific locus in a sample following exposure to the compound of interest to the enrichment of a control-treated sample, wherein a relative chromatin inhibition value greater than two standard deviations from the relative chromatin inhibition value for the control-treated sample indicates that the compound of interest is a modulator of aberrant chromatin accessibility. In particular embodiments, the identified compound reduces aberrant, i.e., abnormal, chromatin accessibility mediated by a protein that induces altered nucleosome positioning and/or depletion at regulatory elements.

In some embodiments, the at least one specific genomic locus binds to chimeric transcription factor EWS-FLI1.

In further embodiments of the present invention, the method includes performing quantitative or real-time polymerase chain reaction (qPCR) on a sample including the nucleosome depleted regions of chromatin.

According to embodiments of the present invention, the relative chromatin inhibition is calculated using the following equation:

where PI and P7 are oncogene-dependent accessible chromatin regions and AURKAIP1 is a region of chromatin that is a positive control.

In particular embodiments, the methods are high-throughput assays as known to those skilled in the art. Briefly, the reagents may be placed in microplates including a grid of small wells typically in multiples of 96. Alternatively, the microplates may be replaced by drops of fluid separated by oil. Utilizing either technique, the samples may be prepared, mixed, incubated, analyzed and/or detected by automation allowing the rapid identification of compounds of interest.

According to embodiments of the present invention, the identified compounds can be used in the diagnosis or treatment of cancers described herein. In particular embodiments, the cancer is Ewing sarcoma.

The general procedure for implementing the methods and assays of the present invention can be readily understood and appreciated by one skilled in the art. Some aspects of the present invention are described in more detail in the following non-limiting Examples. These are not intended to restrict the present invention, and may be modified within the range not deviating from the scope of this invention. EXAMPLES

Example 1

Methods

Cell culture and viability assays

EWS894 and EWS502 cells (Patel et al. 2014) were cultured in RPMI-1640 supplemented with 15% FBS. A673 cells were cultured in RPMI-1640 with 10% FBS. UMRC2 and 786-0 cells were cultured in DMEM with sodium pyruvate, L-glutamine and 4.5 g/L glucose, supplemented with 10% FBS. RPTEC cells were cultured using the REGM™ BulletKit™ (Lonza). HUVEC cells were cultured in the EGM™-2 BulletKit™ (Lonza) supplemented with 10% FBS and maintained at standard growth conditions of 37 °C and 5% C02. Cell viability was assessed by WST-1 (Roche Applied Sciences) according to manufacturer's recommendations. Cells counts were performed using a hemocytometer.

Standard and HT-FAIRE-seq

2 x 10⁷ cells were formaldehyde crosslinked for 7 minutes, quenched with glycine, lysed, and sonicated as previously described (Simon et al. 2012). Five percent of the sonicated material was separated and processed as input control by crosslink reversal, purification by phenol-chloroform extraction, and ethanol precipitation, as previously described (Simon et al. 2012). The remainder of the sonicated lysate was diluted 1 :5 with DNA Binding Buffer and processed through a ChIP DNA clean and concentrate column (Zymo Research #11-379) as per manufacturer's instructions. Quantitative PCR was used to confirm FAIRE DNA enrichment at control genomic loci as described below. For high- throughput sequencing, FAIRE DNA from both replicates was prepared as per manufacturer's recommendations using the TruSeq DNA Sample Prep Kit (Illumina) and 50- bp reads were sequenced (HiSeq 2000, Illumina, UNC High Throughput Sequencing Facility).

Comparison of standard and HT-FAIRE

FAIRE data from seven cell lines (Hl-hESC, HeLa, HepG2, NHEK, K562, GM12878, and HUVEC) were generated previously (Thurman et al. 2012). For all signal- based analyses, one replicate was used for all cell lines except HUVEC, for which data from both replicates were used in parallel. Published sets of FAIRE sites were used in all cases. For HT-FAIRE, data from both replicates were combined, and one set of FAIRE sites was called using MACS2 (Zhang et al. 2008) with a shift-size set to 100. For hierarchical clustering analyses, we computed normalized FAIRE signal in 500-bp non-overlapping windows across the genome. Windows were first filtered for those with an average signal greater than 0.25 (581,514 windows remained) and that fell within an expected range (580,605 windows remained). Windows exhibiting a wide variation across samples (standard deviation greater than 0.5; 9,711 windows remained) were then selected. Signal in these windows was then median-centered and hierarchically clustered using average linkage. ChlP- seq data for histone modifications and transcription factors as well as DNase hypersensitivity were generated previously (Thurman et al. 2012). Repetitive element classes were as defined by RepeatMasker, and genomic redundancy was computed for 36- and 50-bp reads using PeakSeq. (Rozowsky et al. 2009) Motifs in Clusters 1-3 were identified using HOMER (Heinz et al. 2010) using the 500-bp flanking sequence as background. Motifs were considered significant if they had a q-value equal to 0, they occurred in >20% of the target sequences, and had a >3-fold enrichment in the target sequences relative to flanking sequences (background). Motifs in the same transcription factor family were merged for simplicity of presentation.

Quantitative PCR

Input samples were diluted 1 :1000 and FAIRE samples were diluted 1 : 100 in water for comparison of standard and HT-FAIRE. Input samples were diluted 1 : 100 and FAIRE samples used undiluted for the column-based FAIRE screen. Two microliters of each diluted sample was subjected to quantification qPCR in duplicate on the ABI 7900HT using FastStart SYBR Green Master Mix ROX (Roche) in a 10 μΕ final volume. Primer sequences are outlined in Supplementary Table 2. Percent input was determined using the ACt method (Livak and Schmittgen 2001). For the comparison of phenol-chloroform and column-based FAIRE methods, ACt values were normalized to a genomic region near the PRPF31 gene that is negative for FAIRE signal. For the FAIRE screen, relative chromatin inhibition was calculated using the following equation: (((AC

where PI and P7 are EWS-FLIl -dependent open chromatin regions and AURKAIPl is a region of chromatin that consistently has a positive FAIRE signal.

High throughput-FAIRE screen

The automated, high-throughput FAIRE screen was performed in a 96-well format. Compound and vehicle controls were used in the assay at a final concentration of 10 μΜ in 0.1% DMSO. Compounds were plated onto a 96-well V-bottom cell culture plate (Greiner Bio-one #651 180), and EWS894 cells were added using a Multidrop Titertek insrument to a final concentration of 1 x 10⁵ cells per well in a 100 \iL final volume of cell culture media (RPMI supplemented with 15% FBS). Cells were incubated with compound for 16 hours at 37 °C and 5% C0₂ then harvested as follows. A Multidrop Titertek was used to add formaldehyde diluted in cell culture media to a final concentration of 1% per well. Plates were incubated for 5 minutes at 37 °C and 5% C0₂, followed by addition of glycine to a final concentration of 125 mM and incubation at room temperature for 5 minutes. Plates were centrifuged for 5 minutes at 500 x g (Eppendorf 5810R centrifuge) to pellet the cells. Media was removed by quickly inverting the plate. Cells were washed once with phosphate buffered saline (PBS), pelleted as described, then resuspended in 50 μΐ, FAIRE buffer (10 mM Tris, pH 8.0; 2% Triton-X-100; 1% SDS; 100 mM NaCl; 1 mM EDTA). A Tecan Evo 200 was used for all subsequent liquid handling. Cells were suspended in FAIRE buffer to a 0.2 mL 96-raised well PCR plate (Genesee Scientific #27-105) for sonication. The plates were sealed with a 96-well silicone sealing mat (Genesee Scientific #22-513), and a pin lid was pushed through the seal (Matrical Bioscience #SL0096-P21-SS). Plates were sonicated (SonicMan sonicator, Matrical Bioscience) for 20 cycles at 15 seconds at 60% power. Following sonication, 700 U RNase (5 Prime #2900142) was added to each well, and the plates were incubated for 5 minutes at room temperature. Untreated samples from the first column of the plate were removed and pooled for input DNA. The input sample was digested with 20 ^ig proteinase K at 55 °C overnight then purified by phenol-chloroform extraction. Liquid from the remaining wells in columns 2 to 12 was transferred to a ZR-96 ChIP DNA clean and concentrator column (Zymo Research #D5207), and DNA was washed according to manufacturer instructions except that a QiaVAC 96 (Qiagen #19504) was used instead of centrifugation. DNA was eluted from the columns in a 100 L Elution Buffer (Zymo Research). One ^ig of each input sample was separated on a 1.5% agarose gel to confirm fragmentation. Ten μΐ, of the remaining input was diluted 1 : 100 and added back to the plate containing the FAIRE DNA. Buffer alone was also added to the plate as a control for qPCR. Two μΐ, of each sample were transferred from the 96-well plate to a 384-plate for use in qPCR as described above (Supplemental Table 2).

Standard FAIRE quantitative PCR

Cells were treated with 1% formaldehyde followed by inactivation with 125 mM glycine. Cells were resuspended in 2 ml of FAIRE lysis buffer (10 mM Tris-HCl pH 8, 2% Triton X-100, 1% SDS, 100 mM NaCl, 1 mM EDTA) and sonicated (Misonix Sonicator® 3000). Lysates were then subjected to organic extraction using phenol-chloroform to isolate nucleosome-depleted chromatin. Chromatin fragments were incubated with RNaseA for 30 min at 37 °C, proteinase K for 1 hour at 55 °C, followed by overnight crosslink reversal at 65 °C. FAIRE DNA samples were purified (Zymo DNA Clean & Concentrate columns, #11- 379). Input control DNA was treated with RNaseA and proteinaseK prior to organic extraction.

qPCR reagents were combined in a 384- well plate in 10 μΕ reactions containing 5 μΐ_^ of 2X iTaq™ Universal SYBR® Green Supermix (Bio-Rad), 3 iL of 1 μΜ primer pair mix, 1 iL of water, and 1 iL diluted DNA. qPCR was performed using ViiA™ 7 Real-Time PCR system (Applied Biosystems) and each region was analyzed using the ACt method, calculating the FAIRE DNA quantification relative to the corresponding input control. The effect of UNC0621 treatment on each region was determined by ACtuNco6₂i-ACtDMSo- Soft agar colony growth

Cells were suspended in 0.5% low melting point agarose, IX RPMI, 15% fetal bovine serum at a density of 4500 cells per well and layered over one mL of base agar (0.6% agarose, IX RPMI, 15% fetal bovine serum) in a 6-well dish. UNC0621 or DMSO was diluted in top agar layer to desired final concentration. Plates were overlayed with additional RPMI containing compound on day 5 and day 11. Plates were stained with MTT (0.5 mg/ml) on day 15 to visualize cell colonies.

Cell viability assays

Cell viability was assessed by CellTiter-Glo® (Promega) according to manufacturer's recommendations.

Apoptosis

Cells were prepared and stained using BD Pharmingen™ FITC Annexin V Apoptosis Detection Kit and protocol (cat. No. 556547). Flow cytometry was performed immediately after staining.

Western blots

Proteins were extracted using RIPA lysis and separated by SDS-PAGE (Bio-Rad AnyKD™ SDS-PAGE gel, cat. No. 456-9035) then transferred onto nitrocellulose. EWS- FLU was detected using a-Fli antibody (C-19, Santa Cruz sc-356) with tubulin (Sigma T9026) as a loading control. Proteins were detected using fluorescent secondary antibodies, and EWS-FLI1 and tubulin bands were quantified (LiCor) and plotted as a ratio. Example 2

Compound Synthesis of

2-(4-Ethyl-l,4-diazepan-l-yl)-N-(l-isopropylpiperidin-4-yl)-6-methoxy-7-(3-(piperidin- l-yl)propoxy)quinazoIin-4-amine

Conditions and reagents: (a) 1-ethylhomopiperazine, CF₃COOFl, z^'-PrOH, 160 °C, microwave, %.

Chemistry General Procedures

HPLC spectra for all compounds were acquired using an Agilent 6110 Series system with UV detector set to 254 nm. Samples were injected (5 μί) onto an Agilent Eclipse Plus 4.6 x 50 mm, 1.8 μΜ C18 column at room temperature. A linear gradient from 10% to 100% B (MeOH + 0.1% acetic acid) in 5.0 min was followed by pumping 100% B for another 2 minutes with A being H₂0 + 0.1% acetic acid. The flow rate was 1.0 mL/min. Mass spectra (MS) data were acquired in positive ion mode using an Agilent 6110 single quadrupole mass spectrometer with an electrospray ionization (ESI) source. High-resolution mass spectra (HRMS) were acquired using a Thermo LTqFT mass spectrometer under FT control at 100,000 resolution. Nuclear Magnetic Resonance (NMR) spectra were recorded at Varian Mercury spectrometer with 400 MHz for proton (^!H NMR) and 100 MHz for carbon (¹³C NMR); chemical shifts are reported in ppm (δ). Preparative HPLC was performed on Agilent Prep 1200 series with UV detector set to 220 nm. Samples were injected onto a Phenomenex Luna 75 x 30 mm, 5 μτη CI 8 column at room temperature. The flow rate was 30 mL/min. A linear gradient was used with 10% of MeOH (A) in 0.1 % TFA in H₂0 (B) to 100% of MeOH (A). 2-(4-Ethyl-l,4-diazepan-l-yl)-N-(l-isopropylpiperidin-4-yl)-6-methoxy-7-(3-(piperidin- l-yl)propoxy)quinazolin-4-amine). A mixture of the intermediate 2 (75 mg, 0.16 mmol) (prepared from the commercially available methyl-4-hydorxy-3-methoxybenzoate (1) according to the previously reported procedures (Liu et al. 201 1)), 1 -ethylhomopiperazine (41 mg, 0.32 mmol), and TFA (72 mg, 0.64 mmol) in z^'-PrOH (0.26 mL) was heated by microwave irradiation to 160 °C for 15 min in a sealed tube. After concentration in vacuo, the crude product was purified by preparative HPLC with a gradient from 10% of MeOH (A) in 0.1% TFA in H₂0 (B) to 100% of MeOH (A). The resulting product was basified with saturated aq. NaHC0₃ and extracted with CH₂C1₂ to afford the title compound U C0621 (3) as a white solid (71 mg, 80% yield). 1H NMR (400 MHz, CDC1₃) δ 6.87 (s, 1H), 6.72 (s, 1H), 4.98 (d, J = 7.1 Hz, 1H), 4.12 (t, J = 6.8 Hz, 2H), 4.08 - 3.98 (m, 1H), 3.98 - 3.91 (m, 2H), 3.90 - 3.80 (m, 5H), 2.92 - 2.84 (m, 2H), 2.79 - 2.68 (m, 3H), 2.65 - 2.57 (m, 2H), 2.54 (q, J = 7.1 Hz, 2H), 2.43 (t, J= 12 Hz, 2H), 2.40 - 2.23 (m, 6H), 2.21 - 2.10 (m, 2H), 2.08 - 1.91 (m, 4H), 1.61 - 1.48 (m, 6H), 1.45 - 1.36 (m, 2H), 1.13 - 0.98 (m, 9H). ¹³C NMR (100 MHz, CDC1₃) δ 158.73, 158.12, 154.05, 149.80, 145.20, 107.06, 102.78, 101.70, 67.51, 56.76, 55.93, 55.86, 54.66 (2C), 54.58, 54.45, 51.67, 48.77, 47.89 (2C), 46.28, 45.86, 32.76 (2C), 27.98, 26.60, 26.1 1 (2C), 24.57, 18.60 (2C), 12.51. HPLC: 98%; ¾ 0.838 min. MS (ESI): 568 [M + H]⁺. HRMS: calculated for C₃₂H₅₃N₇0₂ [M + H]⁺: 568.4339. Found: 568.4350.

Example 3

Validation of FAIRE using DNA purification columns in place of phenol- chloroform extraction

As currently applied, FAIRE, a biochemical assay for the enrichment of nucleosome- depleted regions of the genome, is dependent on organic extraction with a mixture of phenol and chloroform. Since this extraction step is not easily automated, and screening large chemical libraries requires sufficient throughput, we adapted and miniaturized FAIRE by replacing organic extraction with column-based selection and robotic automation, hereafter termed high throughput FAIRE (HT-FAIRE). Since the chromatin fractionation step is central to this technique, we compared the performance of the methods by qPCR. Both FAIRE methods demonstrated concordant enrichment at a series of promoter and enhancer regions (Supplementary Fig. 1). We then compared the performance of both methods genome-wide. For these studies we used primary human endothelial cells (HUVEC) since these cells have been well studied offering multiple genomic datasets for subsequent integrative analyses of the FAIRE results (ENCODE Tier 2). HT-FAIRE signal mimicked that of standard FAIRE with enrichment at active transcriptional start sites (TSS) that positively correlated with RNA levels, as well as enrichment at CTCF sites, consistent with previously described FAIRE studies (Fig. la-c), (Simon et al. 2012). However, signal enrichment by HT-FAIRE was less robust at TSS, consistent with the qPCR results. Of the top 10,000 nucleosome-depleted regions detected in HUVEC by HT-FAIRE, approximately 90% overlapped sites identified by standard FAIRE in HUVEC. In contrast, fewer than 50% of enriched regions overlapped FAIRE sites from any of six other cell lines (Fig. Id), demonstrating a level of specificity similar to previous reports comparing standard FAIRE between cell types (Song et al. 2011). These data suggest that column and standard FAIRE identify highly similar regions although with some platform-specific disparity.

We then specifically examined those genomic regions that most discriminate column and standard FAIRE and also demonstrate HUVEC cell-type-specificity. Hierarchical clustering of these -9,700 genomic regions identified three groups. Cluster 1 (1,805 regions) showed FAIRE enrichment in all cell lines examined by standard FAIRE but lacked signal in HUVEC HT-FAIRE. Cluster 2 (6,017 regions), by far the largest, consisted of regions with HUVEC-specific signal enrichment detected by both column and standard FAIRE. Cluster 3 (843 regions) was marked by column-FAIRE-specific enrichment (Fig. le). Regions in each cluster were associated with genes (Genome Regions Enrichment of Annotations Tool (GREAT), (McLean et al. 2010)). As expected, the HUVEC-selective sites detected by both HT- and standard FAIRE (Cluster 2) were tightly linked with endothelial cell ontologies (including angiogenesis, q = 4.4 x 10^"12) and regulation of cell-substrate adhesion (q = 6.5 x 10^"7) (Supplementary Fig. 2a). No significant gene ontologies were associated with the regions in clusters specific for either standard or HT-FAIRE (clusters 1 and 3). To further test whether FAIRE-enriched sites represented regulatory elements, we annotated the sites from each cluster with the degree of overlap with active histone modifications (H2A.Z, H3K4mel, H3K4me2, H3K4me3, H3K9ac, H3K27ac, H3K36me3, H3K79me2, H4K20mel), repressive histone modifications (H3K9mel, H3K9me3, H3K27me3), as well as numerous transcription factors (CTCF, RNA Polymerase II, MAX, FOS, JUN, GATA2, FLU, EZH2) assessed by ChlP-seq that are known to be important in endothelial cell biology (Consortium et al. 2012; Patel et al. 2012) (Fig. If). Sites in Cluster 2 (platform-independent, HUVEC-specific) were associated primarily with active histone modifications as well as binding sites for FLU, FOS, JUN, GATA2, and RNA Polymerase II. The prevalence of putative FLU and FOS/JUN binding sites was corroborated by the enrichment of ETS and API DNA sequence motifs in these regions (p < 1 x 10 , Supplementary Fig. 2b). ETS (specifically ETV2) and API factors are both known to play prominent roles in endothelial development (De Val and Black 2009; Meadows et al. 2011). Sites in Cluster 2 were associated with both active and repressive modifications, whereas regions in cluster 1 were more closely associated with repressive modifications and EZH2 binding (Fig. If). We asked whether the difference in sequencing read length between standard FAIRE-seq data (ENCODE, 36-bp reads) and HT- FAIRE-seq data (50-bp reads) could partially account for the variability in mapping (Supplementary Fig. 2c, top) and GC/AT content (Supplementary Fig. 2c, bottom) at Clusters 1 and 3. However, repeating these analyses after truncating the HT-FAIRE sequencing reads to 36 bp did not change the hierarchical clustering or histone modification associations (Supplementary Fig. 2d-e). Clusters 1 and 3 were distinguished by enrichment for repetitive regions, with each cluster associated with a specific repetitive element class: satellites (82% of Cluster 1 sites) and simple repeats (71% of Cluster 3 sites) (Fig. lg and Supplementary Fig. 2f). The basis of their differential enrichment may reflect chromatin variation at these regions that are detected by the biochemical properties specific to organic or solid phase purification. Overall, the high degree of overlap between the two methods, as well as consistent recovery of regulatory elements relevant to endothelial cell biology indicates that the two approaches are experimentally similar and identify regions that are biologically meaningful.

Example 4

Application of HT-FAIRE to a small molecule screen based on a tumor-specific chromatin signature

Following method validation, HT-FAIRE was automated using liquid-handling robotics and a 96-well column-based DNA purification format for the purpose of screening a chromatin-focused compound library. The compound library consisted of 639 small molecules, including those designed to target histone methyltransferases, methyl-lysine reader proteins, histone demethylases, DNA methyltransferases, and acetyl-lysine reader proteins. For the screen, a Ewing Sarcoma patient-derived cell line that grows in suspension culture (EWS894) was exposed to 10 μΜ of each compound for 16 hours. HT-FAIRE was performed (Fig. 2a, Supplementary Methods) followed by quantitative PCR on four regions. Two regions were selected from the Ewing Sarcoma- specific sites, each encompassing a microsatellite repeat that we had previously found was aberrantly accessible in multiple Ewing Sarcoma cell lines with accessibility dependent on EWS-FLI1. Two control regions that consistently demonstrate FAIRE enrichment across many cell types were also tested (Patel et al. 2012); treatment-associated changes at these regions would be indicative of nonspecific activity. Across the range of compounds tested, the test regions demonstrated highly concordant signals (Pearson r = 0.98). This result demonstrates the remarkable consistency achievable by HT-FAIRE and supports combining the values from both sites for subsequent analytics.

To quantify the degree to which chromatin accessibility changed following compound treatment, we calculated a "relative chromatin inhibition" score that compared the relative enrichment at the EWS-FLll targeted regions to the control regions (Supplementary Methods) (Fig. 2b-c). Compounds were considered hits if they had a relative chromatin inhibition value greater than two standard deviations from the average FAIRE signal for vehicle-treated controls (Supplementary Table 1). Sixty-one compounds that reduced chromatin accessibility met these criteria (Fig. 2b and 2c).

We next examined EWS894 cell viability after treatment with each hit compound at 16 and 72 hours. Of the nine compounds that showed the greatest reduction in chromatin accessibility (Supplementary Fig. 3), three compounds significantly affected viability at 72 hours. Compound U C0621 (Fig. 2d) had the lowest EC₅₀ value (456 nM) at 72 hours, and was the only compound out of the 61 hits tested to affect cellular viability at concentrations below 1 μΜ (Supplementary Fig. 3f). We therefore further characterized the effects of UNC0621 treatment on chromatin accessibility and cell viability in Ewing Sarcoma cells.

To ensure that the observed inhibition of chromatin accessibility was not specific to

HT-FAIRE or limited to the regions tested in the screen, we next assayed chromatin accessibility by standard FAIRE followed by qPCR at several EWS-FLll binding sites, including those in the original screen (Patel et al. 2012) (Fig. 2e). Treatment of EWS 894 cells with UNC0621 resulted in a reduction of chromatin accessibility at all eight of the EWS-FLll binding sites tested relative to three unbound control regions. These data confirm the results of the original screen and demonstrate that U C0621 reproducibly affects chromatin accessibility at many EWS -FLU -targeted sites. In addition, these results indicate that UNC0621 acts to decrease FAIRE signal at EWS -FLU -target sites rather than increase signal at the control regions. These data also show that the results obtained from the automated screen were not specific to the column-based chemistry underlying the FAIRE method. Example 5

UNC0621 reverses EWS-FLIl-dependent chromatin accessibility and halts proliferation of Ewing Sarcoma cell lines

Next, we determined whether UNC0621 treatment reduced chromatin accessibility in a dose-dependent fashion. Treatment of EWS894 cells with UNC0621 for 16 hours resulted in a dose-dependent decrease in chromatin accessibility as assayed by HT-FAIRE, with an apparent IC₅₀ between 120 and 370 nM (Fig. 2f). This effect was not observed with a control compound with no structural similarity to UNC0621 that was selected based on the absence of an effect in our screen. The effect of UNC0621 on chromatin accessibility was independent of an effect on cell viability (Supplementary Fig. 3f).

To explore the specificity of the biological activity of UNC0621, we compared the effect of UNC0621 treatment on the proliferation and viability of Ewing Sarcoma cells to other cancer cell lines and primary human cell lines. After 72 hours of treatment, the three Ewing Sarcoma cell lines tested showed a significant decline in cell viability with an EC50 of 0.4 to 1.2 μΜ (Fig. 3a). These concentrations had little effect on the proliferation and survival of two renal carcinoma cell lines (Fig. 3b) and two primary cell types of epithelial and endothelial lineage (human renal proximal tubule epithelial cells (RPTEC) and human umbilical vein endothelial cells (HUVEC)) (Fig. 3c). Increasing cytotoxicity was noted in Ewing Sarcoma cells under continuous treatment for 6 days (Fig. 3d). Thus, Ewing Sarcoma cells are selectively sensitive to UNC0621 treatment.

We then began to explore the biochemical and cellular mechanisms underlying the effect of UNC0621 on Ewing Sarcoma cells. Since the continued presence of EWS-FLI1 is necessary to maintain chromatin state at the target sites, we tested whether EWS-FLI1 protein levels were altered in the presence of UNC0621. No reduction in EWS-FLI1 protein levels was observed following UNC0621 treatment (Supplementary Fig. 4a,b) in support of the hypothesis that the compound acts on a factor that mediates the activity of EWS-FLI1 on chromatin, rather than by affecting EWS-FLI1 protein levels. To test whether UNC0621 affected viability through the induction of apoptosis, we examined Annexin V reactivity by FACS. Surprisingly, treatment of EWS894 cells for 72 hours with UNC0621 was not associated with increased apoptosis (Supplementary Fig. 4c, d). We then tested whether the effects of UNC0621 persisted after withdrawal of the compound. Following exposure to UNC0621 (or control treatment) for three days, identical numbers of viable cells were placed in growth medium lacking compound and counted daily (Fig. 3e). UNC0621 exposure delayed the re-initiation of proliferation by approximately two days. These data indicate that a fraction of EWS894 cells remain viable despite UNC0621 treatment and that treatment results in a persistent but reversible effect on proliferation.

Finally, the influence of UNC0621 on anchorage-independent growth, a characteristic feature of cancer cells was tested. Colony formation in soft agar was inhibited at 200 and 400 nM but not at 100 nM (Fig. 3f, g). Interestingly, the concentrations associated with absence of colony formation had minimal effect on viability in short-term culture. Taken together, these data demonstrate that UNC0621 affects cell viability, proliferation, and transformation in Ewing Sarcoma at nanomolar concentrations.

FAIRE has been adapted and validated as a high-throughput, automated assay for chromatin accessibility. Applying this method to screen a chromatin-focused chemical library enabled the identification of a compound that alters a disease-specific chromatin signature. In contrast to previous efforts to inhibit EWS-FLI1 activity that have focused on gene expression or physical interaction (Stegmaier et al. 2007; Owen et al. 2008; Erkizan et al.

2009; Boro et al. 2012; Kovar 2014; Sankar et al. 2014), this method offers a way to identify therapeutics based on variation in chromatin accessibility, a universal genomic feature determined by transcriptional regulators and chromatin regulatory proteins (Simon et al.

2014).

This high-throughput method is applicable to any disease associated with a measurable change in chromatin accessibility at specific genomic loci. This approach is important as it offers a general strategy to disrupt the function of proteins with structures that are not suitable for small molecule binding by targeting an associated, specific defect in chromatin regulation without the necessity to completely characterize the biochemical pathways and partners involved. The chemical probes identified by this method could be used to elucidate the mechanisms of chromatin dysregulation in disease, lead to the identification of valid molecular targets, and serve as starting points for drug discovery efforts.

Additionally, it is proposed that identified compound, UNC0621, can be used to treat diseases associated with a measurable change in chromatin accessibility at specific genomic loci.

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The foregoing is illustrative of the present invention, and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

What is claimed:

1. A compound having the following structure:

and pharmaceutically acceptable salts thereof.

2. A pharmaceutical composition comprising a compound of claim 1 and a pharmaceutically acceptable carrrier.

3. A pharmaceutical formulation comprising the compound of claim 1, wherein the pharmaceutical formulation is a parenteral formulation.

4. The pharmaceutical formulation of claim 3, wherein the pharmaceutical formulation is an intravenous formulation.

5. The pharmaceutical formulation of claim 3, wherein the pharmaceutical formulation is an intraperitoneal formulation.

6. The pharmaceutical formulation of claim 3, wherein the pharmaceutical formulation is an oral formulation.

7. A method of treating cancer comprising administering an effective amount of a compound of claim 1 to a subject in need thereof.

8. The method of claim 7, wherein the cancer is a peripheral primitive neuroectodermal tumor (pPNET).

9. The method of claim 7, wherein the cancer is one of a Ewing family of tumors

(EFTs).

10. The method of claim 7, wherein the cancer is a Ewing sarcoma.

11. The method of claim 7, wherein the subject is mammalian.

12. The method of claim 11, wherein the subject is human.

13. The method of claim 7, wherein the subject has been diagnosed with Ewing sarcoma or is suspected of having Ewing sarcoma.

14. A method of reducing chromatin accessibility, inhibiting proliferation of a Ewing sarcoma cell, inhibiting transformation of a Ewing sarcoma cell and/or decreasing Ewing sarcoma cell viability, wherein the method comprises contacting a target cell with a compound of claim 1.

15. The method of claim 14, wherein the target cell is a cancer cell.

16. The method of claim 15, wherein the cell is derived from a Ewing sarcoma cell line.

17. The method of claim 15, wherein the cell is a Ewing sarcoma cell.

18. An in vitro method for identifying a compound that modulates aberrant chromatin accessibility at at least one specific genomic locus, the method comprising:

(a) contacting a sample comprising the at least one specific genomic locus with a compound of interest;

(b) contacting the sample with formaldehyde;

(c) obtaining cells from (b) and suspending the cells in a buffer;

(d) sonicating the buffer comprising the suspended cells;

(e) adding a nuclease to the buffer from (d);

(f) subjecting the buffer from (e) to a solid-phase support system;

(g) eluting a nucleic acid from the solid-phase support system; and (h) comparing the enrichment at the at least one specific locus in a sample following exposure to the compound of interest to the enrichment of a control-treated sample wherein a relative chromatin inhibition value greater than two standard deviations from the relative chromatin inhibition value for the control-treated sample indicates that the compound of interest is a modulator of aberrant chromatin accessibility.

19. The method of claim 18 further comprising subjecting at least some of the ehited solution comprising the nucleic acid to quantitative polymerase chain reaction (qPCR).

20. The method of claim 18, wherein relative chromatin inhibition is calculated using the following equation:

(((ACtpi/AC uRKAiPi) + (ACtp₇/ACt_AURKAipi)y2),

where P I and P7 are oncogene-dependent accessible chromatin regions and AURKAIPl is a region of chromatin that is a positive control.

21. The method of claim 18, wherein the cancer is Ewing sarcoma.

22. The method of claim 18, wherein the at least one specific genomic locus binds to EWS-FLI1.

23. The method of claim 18, wherein the sample comprises a Ewing sarcoma cell, a cell from a Ewing sarcoma cell line, or a combination thereof.

24. The method of claim 18, wherein modulation of aberrant chromatin accessibility is a reduction of aberrant chromatin accessibility.

25. The method of claim 18, wherein the assay is a high-throughput assay.