WO2019246571A1 - Compositions and methods for treating adrenocortical carcinoma - Google Patents

Abstract

The present invention features compositions and methods for the treatment of ACC.

Description

COMPOSITIONS AND METHODS FOR TREATING ADRENOCORTICAL

CARCINOMA

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of the following U.S. Provisional Application No.: 62/688,808, filed June 22, 2018, the entire contents of which are incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY

SPONSORED RESEARCH

This invention was made with government support under Grant No.

1R01DK100653A awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Adrenocortical carcinoma (ACC) is a rare endocrine malignancy. When caught early, ACC can be treated successfully with surgery. Late stage cancer is characterized by poor survival. Chemotherapy, radiotherapy and the adrenolytic agent mitotane are used to control, but not cure inoperable or metastatic ACC. Improved therapeutic methods for the treatment of ACC are urgently required.

SUMMARY OF THE INVENTION

As described below, the present invention features compositions and methods for the treatment of ACC.

In one aspect, the invention provides a method of inhibiting cell proliferation in Adrenocortical Carcinoma, the method comprising contacting a cell of the Adrenocortical Carcinoma with an agent that inhibits Fibroblast Growth Factor Receptor 2 (FGFR2) activity.

In another aspect, the invention provides a method for treating Adrenocortical Carcinoma in a subject in need thereof comprising administering to the subject a

therapeutically effective amount of an agent that inhibits Fibroblast Growth Factor Receptor 2 (FGFR2) activity.

In various embodiments of the above aspects, the agent is a small molecule, protein, or polynucleotide. In other embodiments, the agent binds to FGFR2 and inhibits ligand binding. In other embodiments, the agent is a tyrosine kinase inhibitor. In other

BOS 48623703v1 embodiments, the agent is a small molecule inhibitor, an antibody specific for fgfr2, or an antigen-binding fragment of an antibody specific for FGFR2. In other embodiments, the small molecule inhibitor is selected from the group consisting of AZD4547, BGJ398

(infigratinib), Alofanib (RPT835), and SSR128129E; and the protein is Bemarituzumab (FPA144). In other embodiments, the agent inhibits FGFR2 expression. In other

embodiments, the agent is a polynucleotide. In other embodiments, the agent is an inhibitory nucleic acid molecule. In other embodiments, the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA or a vector encoding an inhibitory nucleic acid molecule. In other embodiments, the subject is a mammal (e.g., human). In other

embodiments, the composition is administered intravenously, subcutaneously,

intraperitoneally, orally, via inhalation, or locally. In other embodiments, the administering involves direct administration into and around the adrenal gland.

Compositions and articles defined by the invention were isolated or otherwise manufactured in connection with the examples provided below. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et ah, Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise.

By“Adrenocortical carcinoma (ACC)” is meant a highly malignant endocrine tumor. The diagnosis of malignancy relies on clinical, biological, and imaging features, which may include characterizing one or more of steroid hormone excess, proliferative index, mitotic count, and Ki67 index.

By“Fibroblast Growth Factor Receptor 2 (FGFR2) polypeptide” is meant a cell surface receptor for fibroblast growth factor or a fragment thereof having tyrosine kinase activity and having at least about 85% or greater amino acid sequence identity to Uniprot Identifier P21802-1. An exemplary FGFR2 amino acid sequence is provided below: >sp | P21802 | FGFR2 HUMAN Fibroblast growth factor receptor 2 OS=Homo sapiens OX=9606 GN=FGFR2^_PE=1 SV=1

MVSWGRFICLVWTMATLSLARPSFSLVEDTTLEPEEPPTKYQISQPEVYVAAPGESLEV

RCLLKDAAVISWTKDGVHLGPNNRTVLIGEYLQIKGATPRDSGLYACTASRTVDSETWYF

MVNVTDAISSGDDEDDTDGAEDFVSENSNNKRAPYWTNTEKMEKRLHAVPAANTVKFRCP

AGGNPMPTMRWLKNGKEFKQEHRIGGYKVRNQHWSLIMESWPSDKGNYTCWENEYGSI

NHTYHLDWERSPHRPILQAGLPAASTWGGDVEFVCKVYSDAQPHIQWIKHVEKNGSK

YGPDGLPYLKVLKAAGWTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFHSAWLTVL

PAPGREKEITASPDYLEIAIYCIGVFLIACMWTVILCRMKNTTKKPDFSSQPAVHKLTK

RIPLRRQVTVSAESSSSMNSNTPLVRITTRLSSTADTPMLAGVSEYELPEDPKWEFPRDK

LTLGKPLGEGCFGQWMAEAVGIDKDKPKEAVTVAVKMLKDDATEKDLSDLVSEMEMMKM

IGKHKNIINLLGACTQDGPLYVIVEYASKGNLREYLRARRPPGMEYSYDINRVPEEQMTF

KDLVSCTYQLARGMEYLASQKCIHRDLAARNVLVTENNVMKIADFGLARDINNIDYYKKT

TNGRLPVKWMAPEALFDRVYTHQSDVWSFGVLMWEIFTLGGSPYPGIPVEELFKLLKEGH

RMDKPANCTNELYMMMRDCWHAVPSQRPTFKQLVEDLDRILTLTTNEEYLDLSQPLEQYS

PSYPDTRSSCSSGDDSVFSPDPMPYEPCLPQYPHINGSVKT

By“Fibroblast Growth Factor Receptor 2 (FGFR2) polynucleotide” is meant a nucleic acid sequence encoding an FGFR2 polypeptide or fragment thereof. An exemplary FGFR2 nucleic acid sequence is provided at NM_000l4l.4, which is reproduced below:

1 ggcggcggct ggaggagagc gcggtggaga gccgagcggg cgggcggcgg gtgcggagcg

61 ggcgagggag cgcgcgcggc cgccacaaag ctcgggcgcc gcggggctgc atgcggcgta 121 cctggcccgg cgcggcgact gctctccggg ctggcggggg ccggccgcga gccccggggg 181 ccccgaggcc gcagcttgcc tgcgcgctct gagccttcgc aactcgcgag caaagtttgg 241 tggaggcaac gccaagcctg agtcctttct tcctctcgtt ccccaaatcc gagggcagcc 301 cgcgggcgtc atgcccgcgc tcctccgcag cctggggtac gcgtgaagcc cgggaggctt 361 ggcgccggcg aagacccaag gaccactctt ctgcgtttgg agttgctccc cgcaaccccg 421 ggctcgtcgc tttctccatc ccgacccacg cggggcgcgg ggacaacaca ggtcgcggag 481 gagcgttgcc attcaagtga ctgcagcagc agcggcagcg cctcggttcc tgagcccacc 541 gcaggctgaa ggcattgcgc gtagtccatg cccgtagagg aagtgtgcag atgggattaa 601 cgtccacatg gagatatgga agaggaccgg ggattggtac cgtaaccatg gtcagctggg 661 gtcgtttcat ctgcctggtc gtggtcacca tggcaacctt gtccctggcc cggccctcct 721 tcagtttagt tgaggatacc acattagagc cagaagagcc accaaccaaa taccaaatct 781 ctcaaccaga agtgtacgtg gctgcgccag gggagtcgct agaggtgcgc tgcctgttga 841 aagatgccgc cgtgatcagt tggactaagg atggggtgca cttggggccc aacaatagga 901 cagtgcttat tggggagtac ttgcagataa agggcgccac gcctagagac tccggcctct 961 atgcttgtac tgccagtagg actgtagaca gtgaaacttg gtacttcatg gtgaatgtca 1021 cagatgccat ctcatccgga gatgatgagg atgacaccga tggtgcggaa gattttgtca 1081 gtgagaacag taacaacaag agagcaccat actggaccaa cacagaaaag atggaaaagc 1141 ggctccatgc tgtgcctgcg gccaacactg tcaagtttcg ctgcccagcc ggggggaacc 1201 caatgccaac catgcggtgg ctgaaaaacg ggaaggagtt taagcaggag catcgcattg 1261 gaggctacaa ggtacgaaac cagcactgga gcctcattat ggaaagtgtg gtcccatctg 1321 acaagggaaa ttatacctgt gtagtggaga atgaatacgg gtccatcaat cacacgtacc 1381 acctggatgt tgtggagcga tcgcctcacc ggcccatcct ccaagccgga ctgccggcaa 1441 atgcctccac agtggtcgga ggagacgtag agtttgtctg caaggtttac agtgatgccc 1501 agccccacat ccagtggatc aagcacgtgg aaaagaacgg cagtaaatac gggcccgacg 1561 ggctgcccta cctcaaggtt ctcaaggccg ccggtgttaa caccacggac aaagagattg 1621 aggttctcta tattcggaat gtaacttttg aggacgctgg ggaatatacg tgcttggcgg 1681 gtaattctat tgggatatcc tttcactctg catggttgac agttctgcca gcgcctggaa 1741 gagaaaagga gattacagct tccccagact acctggagat agccatttac tgcatagggg 1801 tcttcttaat cgcctgtatg gtggtaacag tcatcctgtg ccgaatgaag aacacgacca 1861 agaagccaga cttcagcagc cagccggctg tgcacaagct gaccaaacgt atccccctgc 1921 ggagacaggt aacagtttcg gctgagtcca gctcctccat gaactccaac accccgctgg 1981 tgaggataac aacacgcctc tcttcaacgg cagacacccc catgctggca ggggtctccg 2041 agtatgaact tccagaggac ccaaaatggg agtttccaag agataagctg acactgggca 2101 agcccctggg agaaggttgc tttgggcaag tggtcatggc ggaagcagtg ggaattgaca 2161 aagacaagcc caaggaggcg gtcaccgtgg ccgtgaagat gttgaaagat gatgccacag

2221 agaaagacct ttctgatctg gtgtcagaga tggagatgat gaagatgatt gggaaacaca

2281 agaatatcat aaatcttctt ggagcctgca cacaggatgg gcctctctat gtcatagttg

2341 agtatgcctc taaaggcaac ctccgagaat acctccgagc ccggaggcca cccgggatgg

2401 agtactccta tgacattaac cgtgttcctg aggagcagat gaccttcaag gacttggtgt

2461 catgcaccta ccagctggcc agaggcatgg agtacttggc ttcccaaaaa tgtattcatc

2521 gagatttagc agccagaaat gttttggtaa cagaaaacaa tgtgatgaaa atagcagact

2581 ttggactcgc cagagatatc aacaatatag actattacaa aaagaccacc aatgggcggc

2641 ttccagtcaa gtggatggct ccagaagccc tgtttgatag agtatacact catcagagtg

2701 atgtctggtc cttcggggtg ttaatgtggg agatcttcac tttagggggc tcgccctacc

2761 cagggattcc cgtggaggaa ctttttaagc tgctgaagga aggacacaga atggataagc

2821 cagccaactg caccaacgaa ctgtacatga tgatgaggga ctgttggcat gcagtgccct

2881 cccagagacc aacgttcaag cagttggtag aagacttgga tcgaattctc actctcacaa

2941 ccaatgagga atacttggac ctcagccaac ctctcgaaca gtattcacct agttaccctg

3001 acacaagaag ttcttgttct tcaggagatg attctgtttt ttctccagac cccatgcctt

3061 acgaaccatg ccttcctcag tatccacaca taaacggcag tgttaaaaca tgaatgactg

3121 tgtctgcctg tccccaaaca ggacagcact gggaacctag ctacactgag cagggagacc

3181 atgcctccca gagcttgttg tctccacttg tatatatgga tcagaggagt aaataattgg

3241 aaaagtaatc agcatatgtg taaagattta tacagttgaa aacttgtaat cttccccagg

3301 aggagaagaa ggtttctgga gcagtggact gccacaagcc accatgtaac ccctctcacc

3361 tgccgtgcgt actggctgtg gaccagtagg actcaaggtg gacgtgcgtt ctgccttcct

3421 tgttaatttt gtaataattg gagaagattt atgtcagcac acacttacag agcacaaatg

3481 cagtatatag gtgctggatg tatgtaaata tattcaaatt atgtataaat atatattata

3541 tatttacaag gagttatttt ttgtattgat tttaaatgga tgtcccaatg cacctagaaa

3601 attggtctct ctttttttaa tagctatttg ctaaatgctg ttcttacaca taatttctta

3661 attttcaccg agcagaggtg gaaaaatact tttgctttca gggaaaatgg tataacgtta

3721 atttattaat aaattggtaa tatacaaaac aattaatcat ttatagtttt ttttgtaatt

3781 taagtggcat ttctatgcag gcagcacagc agactagtta atctattgct tggacttaac

3841 tagttatcag atcctttgaa aagagaatat ttacaatata tgactaattt ggggaaaatg

3901 aagttttgat ttatttgtgt ttaaatgctg ctgtcagacg attgttctta gacctcctaa

3961 atgccccata ttaaaagaac tcattcatag gaaggtgttt cattttggtg tgcaaccctg

4021 tcattacgtc aacgcaacgt ctaactggac ttcccaagat aaatggtacc agcgtcctct

4081 taaaagatgc cttaatccat tccttgagga cagaccttag ttgaaatgat agcagaatgt

4141 gcttctctct ggcagctggc cttctgcttc tgagttgcac attaatcaga ttagcctgta

4201 ttctcttcag tgaattttga taatggcttc cagactcttt ggcgttggag acgcctgtta

4261 ggatcttcaa gtcccatcat agaaaattga aacacagagt tgttctgctg atagttttgg

4321 ggatacgtcc atctttttaa gggattgctt tcatctaatt ctggcaggac ctcaccaaaa

4381 gatccagcct catacctaca tcagacaaaa tatcgccgtt gttccttctg tactaaagta

4441 ttgtgttttg ctttggaaac acccactcac tttgcaatag ccgtgcaaga tgaatgcaga

4501 ttacactgat cttatgtgtt acaaaattgg agaaagtatt taataaaacc tgttaatttt

4561 tatactgaca ataaaaatgt ttctacagat attaatgtta acaagacaaa ataaatgtca

4621 cgcaacttat ttttttaata aaaaaaaaaa aaaa

By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

By“ameliorate” is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease (e.g., adrenocortical carcinoma).

By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels. " By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally-occurring polypeptide, while having certain biochemical

modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes,"

"including," and the like; "consisting essentially of' or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

“Detect” refers to identifying the presence, absence or amount of the analyte to be detected.

By“disease” is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Examples of diseases include Adrenocortical carcinoma (ACC).

By "effective amount" is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. In one embodiment, the amount is sufficient to reduce or stabilize cell proliferation or reduce the survival of a cancer cell. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

"Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.

By "inhibitory nucleic acid" is meant a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.

The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different modifications may give rise to different isolated proteins, which can be separately purified.

By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence. By an "isolated polypeptide" is meant a polypeptide of the invention that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the invention. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By“marker” is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder. In one embodiment, aldosterone is a marker.

As used herein,“obtaining” as in“obtaining an agent” includes synthesizing, purchasing, or otherwise acquiring the agent.

By“reduces” is meant a negative alteration of at least 10%, 25%, 50%, 75%, or

100%.

By“reference” is meant a standard or control condition.

A "reference sequence" is a defined sequence used as a basis for sequence

comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or therebetween.

By "siRNA" is meant a double stranded RNA. Optimally, an siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3' end. These dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream. Such siRNAs are used to downregulate mRNA levels or promoter activity. By "specifically binds" is meant a compound or antibody that recognizes and binds a polypeptide of the invention, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the invention.

Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the invention include any nucleic acid molecule that encodes a polypeptide of the invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having“substantial identity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule.

By "hybridize" is meant pair to form a double-stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

For example, stringent salt concentration will ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30° C, more preferably of at least about 37° C, and most preferably of at least about 42° C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30° C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37° C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42° C in 250 mM NaCl, 25 mM trisodium citrate,

1% SDS, 50% formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25° C, more preferably of at least about 42° C, and even more preferably of at least about 68° C. In a preferred embodiment, wash steps will occur at 25° C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 42 C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps will occur at 68° C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS.

Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705,

BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or 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. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e ³ and e ¹⁰⁰ indicating a closely related sequence.

By "subject" is meant a mammal, including, but not limited to, a human or non human mammal, such as a bovine, equine, canine, ovine, or feline.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,

41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms“treat,” treating,”“treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term“about” is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a schematic representation of the Cre-lox system for specific deletion of DNA sequences in the adrenal. The Cre recombinase expression is restricted to the zona glomerulosa (zG).

FIG. 2 - Combined deletion of Trp53 and stabilization of b-catenin results in neoplasia in the mouse adrenal cortex. Histological analysis and immunostaining for B- catenin in adrenal glands from 1, 3 and 5 months mice with adrenal-specific Trp53 deletion (Trp53 KO), b-catenin gain-of-function (Bcat-GOF) or Trp53 deletion and b-catenin gain-of- function (Trp53 KO Bcat-GOF). WT=Wild Type.

FIG. 3 shows that tumor formation is fully penetrant in Trp53 KO Bcat-GOF mice. The graphs at the top of the figure show the percentage of tumor free mice as a function of age. The photographs below show the presence of ACC (right, first panel) in adrenal glands vs. normal control adrenal glands (left, first panel), as well as a photomicrograph of a section of the ACC. The inset photograph and photomicrograph to the right shows the presence of metastases in lung.

FIG. 4 includes four box plots showing hormonal activity Trp53 deletion and b- catenin gain-of-function (Trp53 KO Bcat-GOF) vs. control mice.

FIG. 5 includes two graphs showing changes in FGFR2 isoform expression in b- catenin gain-of-function (Bcat-GOF) or Trp53 deletion and b-catenin gain-of-function (Trp53 KO Bcat-GOF) vs. control mice.

FIG. 6 presents immunohistochemistry results comparing b GOF adrenals with and without treatment with the indicated FGFR2 inhibitor and histogram plots depicting these results.

FIG. 7 provides a series of graphs showing gene expression of EZh2 in b-catenin gain-of-function (Bcat-GOF) or Trp53 deletion and b-catenin gain-of-function (Trp53 KO Bcat-GOF) vs. wild-type control mice at various ages.

FIG. 8 provides micrographs showing that loss of FGFR2 affects zona glomerulosa (zG) identity.

FIGs. 9A and 9B illustrate that FGFR inhibitors impair zona glomerulosa function. FIG. 9A is a plot showing urinary aldosterone levels over time in adult mice treated with AZD4547 and mice treated with vehicle only. The arrow denotes the start of treatments. denotes p < 0.01 and“*” denotes p < 0.05. FIG. 9B, is a graph showing the fold change in Cypl lb2 and Cyclin Dl transcript levels in adrenals from untreated mice and mice treated with BGJ398 for 7 days. “*” denotes p < 0.05. Error bars, denote the mean ± SEM. FIG. 10 provides a series of images illustrating that Fgfr2 deletion prevents zona glomerulosa hyperplasia driven by b-catenin gain-of-function. Representative images of GFP, DAP I, and Dab2 co-immunostaining of control (ASCre/+; mTmG), PC at GOF

(ASCre/+; mTmG; b-catenin fl(ex3)/+), and pCat GOF; Fgfr2 cKO (ASCre/+; mTmG; b- catenin fl(ex3)/+; Fgfr2 fl/fl) adult mice adrenals. Bars denote 50 pm.

DETAILED DESCRIPTION OF THE INVENTION

As described below, the present invention features compositions and methods for the treatment of ACC.

The invention is based, at least in part, on the discovery that FGFR2 receptor antagonists are useful for reducing cell proliferation and treating ACC.

Dysregulation of WNT/p-catenin signaling and TP 53 mutations have been implicated in both sporadic and familial forms of adrenocortical carcinoma (ACC). In fact, genomic characterization has revealed that these pathways are the most frequently altered in ACCs with the poorest outcomes. Despite their critical role in the development and prognosis of ACC, it remains unclear whether the combination of Trp53 loss and b-catenin gain-of- function mutations can efficiently initiate a neoplastic process. To investigate this, the Cypllb2(AS)^Cre mouse model was utilized to generate transgenic mice with adrenal-specific (1) Trp53 deletion ( Trp53^Floxed ), (2) b-catenin gain-of-function (Ctnnb l^FloxedEx3), or (3) Trp53 deletion and b-catenin gain-of-function ( Trp53^Floxed::Ctnnbl^{FloxedEx 3}). Analysis of mice with targeted deletion of Trp53 at 1, 3 and 5 months of age showed increased adrenal weight, but no gross morphological or histological changes compared with controls. Gene expression analysis of whole adrenals showed decreased levels of p27, Ax in 2 and Shh. These findings indicate that Trp53 loss leads to a decrease in the p2l tumor suppressor, as well as a down- regulation of canonical Wnt signaling and progenitor cell activity. These results also indicate that a secondary event is required to override the suppression of Wnt signaling, a key feature of ACC, to promote neoplasia. Alterations in gene expression are shown at Figure 7 in mice of varying ages having b-catenin gain-of-function (ftcat-GOF) or Trp53 deletion and b- catenin gain-of-function (Trp53 KO ftcat-GOF).

Analysis of mice with targeted b-catenin gain-of-function at 1 and 3 months of age showed an expansion of the zona glomerulosa (zG), at the expense of the zona fasciculata (zF), without an overall increase in adrenal size compared with controls. Gene expression analysis showed increased expression of Axin2 and Shh , as well as the ACC-related gene Ezh2. These results suggest that activation of Wnt signaling alone is not sufficient to drive neoplastic transformation. Finally, analysis of mice with the combined Trp53 and b-catenin mutations showed a dramatic expansion of the zG, compared with b-catenin gain-of-function mice, at 1 month of age, and enlarged, hyperplastic adrenals, with focal nodularity at 3 months of age. Gene expression analysis showed elevated expression of Axin2, Shh and Ezh2, but low levels of p21. These results indicate that the combination of Trp53 deletion and b- catenin gain-of-function is sufficient to initiate neoplastic changes in the adrenal cortex. Taken together, our preliminary data show that combining the two most frequently dysregulated pathways in ACC results in marked neoplastic transformation, which may recapitulate the early events in ACC tumorigenesis. In addition, the inventors have discovered increased expression of Fibroblast growth factor receptor 2 (Fgfr2) in bCat-GOF adrenals. Genetic deletion of Fgfr2 in the adult adrenal results in disrupted zG morphology and impaired aldosterone production. In addition, treatment of mice with pharmacological antagonists of FGFR signaling results in a marked decrease in zG proliferation. Accordingly, pharmacological antagonists of FGFR signaling are useful for the treatment of ACC.

Adrenocortical Carcinoma (ACC)

ACC is associated to Li-Fraumeni and Beckwith-Wiedemann syndromes, due to germline TP53 mutations and alterations of the insulin-like growth factor IGF2, respectively. Moreover, there are evidences associating ACC with adult patients with multiple endocrine neoplasia (MEN1), familial adenomatous polyposis coli (FAP) and neurofibromatosis type 1 (NF1). Recently, genomic characterization of ACC has shed light on several genes as potential drivers involved in sporadic adrenocortical tumorigenesis and multiple genomic profiling efforts have confirmed the high prevalence of Wnt/B-catenin and TP53/Rb pathways deregulation as well as IGF2 gene overexpression in the majority of ACC patients. Gain-of- function CTNNB1 mutations and inactivating ZNRF3 (a negative Wnt pathway regulator) mutations or deletions are the most common somatic alterations that leads to a constitutively activation of Wnt signaling in ACC; on the other hand, TP53 loss-of-function constitute the most recurrent genetic alteration associated with the disruption of the TP53/Rb pathway. In fact, clustering analyses showed that p53/Rb and WNT^-catenin pathways were the leading altered pathways in ACCs with poorest outcome.

b-catenin expression and activity is restricted to outer cortical ceils that correspond to the zona glomerulosa. Targeted disruption of beta-catenin in the adrenal cortex impairs its development and maintenance indicating crucial roles for beta-catenin in both embryonic development and in maintenance of the adult organ. Interestingly, despite the frequency of activating b-catenin mutation in the human ACC its adrenocortical specific activation alone is not sufficient to induce a malignant progression in mouse models, although this alteration was able to trigger the development of benign adrenal cortex tumors. Notably, even the combination of b-catenin/WNT activation along with IGF2 overexpression failed to induce ACC in mouse models^{10 11}. These findings suggest that others genetic alterations may act cooperatively with b-catenin/WNT activation to induce malignant tumor progression in the adrenal cortex.

p53 is considered the most important tumor suppressor gene within the adrenal cortex. It acts as a potent transcription factor, playing a key role in maintaining genomic stability. Germline TP53 mutations results in the Li Fraumeni Syndrome (LFS), a cancer predisposition syndrome associated with ACC. The ACC high rates among p53-mutation carriers suggests a tissue-specific manifestation of p53 deficiency. In accordance with this idea, a lO-fold increased incidence of adrenocortical carcinoma had been observed in South- Eastern Brazil where a common germline mutation within the oligomerization domain of p53, p.R337H, has been found in that population. It remains to be determined whether TP53 plays a role in the adrenal cortex homeostasis however Trp53 knockout mice failed to develop ACC, arguing that ACC formation requires mutations in addition to Trp53 loss in the adrenal.

The presence of recurrent mutations in TP53 as well as in genes involved in the WNT signaling in human ACC provides evidence that these genetic alterations may act cooperatively to induce ACC formation. In order to test this hypothesis, the inventors combined the Trp53 loss and B-catenin gain-of-function in the mouse adrenal cortex. First, the inventors investigated the individual tumorigenic role of Trp53 deletion and B-catenin gain-of-function and in the adrenal cortex. Targeted deletion of Trp53 leads to increased adrenal weight but no gross morphological or histological changes associated with a malignant phonotype. In contrast, mice with targeted b-catenin gain-of-function showed an expansion of the zona glomerulosa (zG), without an overall increase in adrenal size compared with controls. Finally, analysis of mice with the combined Trp53 and b-catenin mutations showed a dramatic expansion of the zG, compared with b-catenin gain-of-function mice, hyperplastic adrenals, with focal nodularity These results indicate that the combination of Trp53 deletion and b-catenin gain-of-function is sufficient to initiate neoplastic changes in the adrenal cortex. Therefore, the inventors’ data show that combining the two most frequently dysregulated pathways in ACC results in marked neoplastic transformation, which may recapitulate the early events in ACC tumorigenesis.

Inhibitory Nucleic Acids

Inhibitory nucleic acid molecules are those oligonucleotides that inhibit the expression or activity of an FGFR2 polypeptide. Such oligonucleotides include single and double stranded nucleic acid molecules (e.g., DNA, RNA, and analogs thereof) that bind a nucleic acid molecule that encodes an FGFR2 polypeptide (e.g., antisense molecules, siRNA, shRNA) as well as nucleic acid molecules that bind directly to the polypeptide to modulate its biological activity (e.g., aptamers).

siRNA

Short twenty-one to twenty-five nucleotide double-stranded RNAs are effective at down-regulating gene expression (Zamore et al., Cell 101 : 25-33; Elbashir et al., Nature 411 : 494-498, 2001, hereby incorporated by reference). The therapeutic effectiveness of an siRNA approach in mammals was demonstrated in vivo by McCaffrey et al. (Nature 418: 38- 39.2002).

Given the sequence of a target gene, siRNAs may be designed to inactivate that gene. Such siRNAs, for example, could be administered directly to an affected tissue, or administered systemically. The nucleic acid sequence of a gene can be used to design small interfering RNAs (siRNAs). The 21 to 25 nucleotide siRNAs may be used, for example, as therapeutics to treat FSHD.

The inhibitory nucleic acid molecules of the present invention may be employed as double-stranded RNAs for RNA interference (RNAi)-mediated knock-down of expression. RNAi is a method for decreasing the cellular expression of specific proteins of interest (reviewed in Tuschl, Chembiochem 2:239-245, 2001; Sharp, Genes & Devel. 15:485-490, 2000; Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225-232, 2002; and Hannon, Nature 418:244-251, 2002). The introduction of siRNAs into cells either by transfection of dsRNAs or through expression of siRNAs using a plasmid-based expression system is increasingly being used to create loss-of-function phenotypes in mammalian cells.

In one embodiment of the invention, a double-stranded RNA (dsRNA) molecule is made that includes between eight and nineteen consecutive nucleobases of a nucleobase oligomer of the invention. The dsRNA can be two distinct strands of RNA that have duplexed, or a single RNA strand that has self-duplexed (small hairpin (sh)RNA). Typically, dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleobases) if desired. dsRNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription). Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296:550-553, 2002; Paddison et al. Genes & Devel. 16:948-958, 2002. Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047- 6052, 2002; Miyagishi et al. Nature Biotechnol. 20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505 2002, each of which is hereby incorporated by reference.

Small hairpin RNAs (shRNAs) comprise an RNA sequence having a stem-loop structure. A "stem -loop structure" refers to a nucleic acid having a secondary structure that includes a region of nucleotides which are known or predicted to form a double strand or duplex (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion). The term "hairpin" is also used herein to refer to stem-loop structures. Such structures are well known in the art and the term is used consistently with its known meaning in the art. As is known in the art, the secondary structure does not require exact base-pairing. Thus, the stem can include one or more base mismatches or bulges. Alternatively, the base-pairing can be exact, i.e. not include any mismatches. The multiple stem-loop structures can be linked to one another through a linker, such as, for example, a nucleic acid linker, a miRNA flanking sequence, other molecule, or some combination thereof.

As used herein, the term "small hairpin RNA" includes a conventional stem-loop shRNA, which forms a precursor miRNA (pre-miRNA). While there may be some variation in range, a conventional stem -loop shRNA can comprise a stem ranging from 19 to 29 bp, and a loop ranging from 4 to 30 bp. "shRNA" also includes micro-RNA embedded shRNAs (miRNA-based shRNAs), wherein the guide strand and the passenger strand of the miRNA duplex are incorporated into an existing (or natural) miRNA or into a modified or synthetic (designed) miRNA. In some instances the precursor miRNA molecule can include more than one stem-loop structure. MicroRNAs are endogenously encoded RNA molecules that are about 22-nucleotides long and generally expressed in a highly tissue- or developmental- stage-specific fashion and that post-transcriptionally regulate target genes. More than 200 distinct miRNAs have been identified in plants and animals. These small regulatory RNAs are believed to serve important biological functions by two prevailing modes of action: (1) by repressing the translation of target mRNAs, and (2) through RNA interference (RNAi), that is, cleavage and degradation of mRNAs. In the latter case, miRNAs function analogously to small interfering RNAs (siRNAs). Thus, one can design and express artificial miRNAs based on the features of existing miRNA genes.

shRNAs can be expressed from DNA vectors to provide sustained silencing and high yield delivery into almost any cell type. In some embodiments, the vector is a viral vector. Exemplary viral vectors include retroviral, including lentiviral, adenoviral, baculoviral and avian viral vectors, and including such vectors allowing for stable, single-copy genomic integrations. Retroviruses from which the retroviral plasmid vectors can be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. A retroviral plasmid vector can be employed to transduce packaging cell lines to form producer cell lines. Examples of packaging cells which can be transfected include, but are not limited to, the PE501, PA317, R-2, R-AM, PA12, T19-14c, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy 1 :5-14 (1990), which is incorporated herein by reference in its entirety. The vector can transduce the packaging cells through any means known in the art. A producer cell line generates infectious retroviral vector particles which include polynucleotide encoding a DNA replication protein. Such retroviral vector particles then can be employed, to transduce eukaryotic cells, either in vitro or in vivo. The transduced eukaryotic cells will express a DNA replication protein.

Catalytic RNA molecules or ribozymes that include an antisense sequence of the present invention can be used to inhibit expression of a nucleic acid molecule in vivo (e.g., a nucleic acid molecule encoding an FGFR2 polypeptide). The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et ak, Nature 334:585-591. 1988, and ET.S. Patent Application Publication No. 2003/0003469 Al, each of which is incorporated by reference.

Accordingly, the invention also features a catalytic RNA molecule that includes, in the binding arm, an antisense RNA having between eight and nineteen consecutive nucleobases. In preferred embodiments of this invention, the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif. Examples of such hammerhead motifs are described by Rossi et ak, Aids Research and Human Retroviruses, 8: 183, 1992. Example of hairpin motifs are described by Hampel et ak, "RNA Catalyst for Cleaving Specific RNA Sequences," filed Sep. 20, 1989, which is a continuation-in-part of ET.S. Ser. No. 07/247,100 filed Sep. 20, 1988, Hampel and Tritz, Biochemistry, 28:4929, 1989, and Hampel et al., Nucleic Acids Research, 18: 299, 1990. These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

Essentially any method for introducing a nucleic acid construct into cells can be employed. Physical methods of introducing nucleic acids include injection of a solution containing the construct, bombardment by particles covered by the construct, soaking a cell, tissue sample or organism in a solution of the nucleic acid, or electroporation of cell membranes in the presence of the construct. A viral construct packaged into a viral particle can be used to accomplish both efficient introduction of an expression construct into the cell and transcription of the encoded shRNA. Other methods known in the art for introducing nucleic acids to cells can be used, such as lipid-mediated carrier transport, chemical mediated transport, such as calcium phosphate, and the like. Thus the shRNA-encoding nucleic acid construct can be introduced along with components that perform one or more of the following activities: enhance RNA uptake by the cell, promote annealing of the duplex strands, stabilize the annealed strands, or otherwise increase inhibition of the target gene.

For expression within cells, DNA vectors, for example plasmid vectors comprising either an RNA polymerase II or RNA polymerase III promoter can be employed. Expression of endogenous miRNAs is controlled by RNA polymerase II (Pol II) promoters and in some cases, shRNAs are most efficiently driven by Pol II promoters, as compared to RNA polymerase III promoters (Dickins et al., 2005, Nat. Genet. 39: 914-921). In some embodiments, expression of the shRNA can be controlled by an inducible promoter or a conditional expression system, including, without limitation, RNA polymerase type II promoters. Examples of useful promoters in the context of the invention are tetracycline- inducible promoters (including TRE-tight), IPTG-inducible promoters, tetracycline transactivator systems, and reverse tetracycline transactivator (rtTA) systems. Constitutive promoters can also be used, as can cell- or tissue-specific promoters. Many promoters will be ubiquitous, such that they are expressed in all cell and tissue types. A certain embodiment uses tetracycline-responsive promoters, one of the most effective conditional gene expression systems in in vitro and in vivo studies. See International Patent Application PCT/US2003/030901 (Publication No. WO 2004-029219 A2) and Fewell et al., 2006, Drug Discovery Today 11 : 975-982, for a description of inducible shRNA.

Delivery of Polynucleotides

Naked polynucleotides, or analogs thereof, are capable of entering mammalian cells and inhibiting expression of a gene of interest. Nonetheless, it may be desirable to utilize a formulation that aids in the delivery of oligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference).

Oligonucleotides and other Nucleobase Oligomers

At least two types of oligonucleotides induce the cleavage of RNA by RNase H: polydeoxynucleotides with phosphodi ester (PO) or phosphorothioate (PS) linkages.

Although 2'-OMe-RNA sequences exhibit a high affinity for RNA targets, these sequences are not substrates for RNase H. A desirable oligonucleotide is one based on 2'-modified oligonucleotides containing oligodeoxynucleotide gaps with some or all internucleotide linkages modified to phosphorothioates for nuclease resistance. The presence of

methylphosphonate modifications increases the affinity of the oligonucleotide for its target RNA and thus reduces the IC50. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present invention may be used in conjunction with any technologies that may be developed, including covalently-closed multiple antisense (CMAS) oligonucleotides (Moon et al., Biochem J. 346:295-303, 2000; PCT Publication No. WO 00/61595), ribbon-type antisense (RiAS) oligonucleotides (Moon et al., J. Biol. Chem. 275:4647-4653, 2000; PCT Publication No.

WO 00/61595), and large circular antisense oligonucleotides (U.S. Patent Application Publication No. US 2002/0168631 Al).

As is known in the art, a nucleoside is a nucleobase-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside.

For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2', 3' or 5' hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric structure can be further joined to form a circular structure; open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3' to 5' phosphodiester linkage.

Specific examples of preferred nucleobase oligomers useful in this invention include oligonucleotides containing modified backbones or non-natural intemucleoside linkages. As defined in this specification, nucleobase oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, modified oligonucleotides that do not have a phosphorus atom in their intemucleoside backbone are also considered to be nucleobase oligomers.

Nucleobase oligomers that have modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,

thionoalkylphosphonates, thionoalkylphosphotriest- ers, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of these, and those having inverted polarity, wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms are also included. Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference.

Nucleobase oligomers having modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl intemucleoside linkages, mixed heteroatom and alkyl or cycloalkyl intemucleoside linkages, or one or more short chain heteroatomic or heterocyclic intemucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyl eneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH.sub.2 component parts. Representative United States patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Pat. Nos.

5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216, 141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.

In other nucleobase oligomers, both the sugar and the intemucleoside linkage, i.e., the backbone, are replaced with novel groups. The nucleobase units are maintained for hybridization with a gene encoding an FGFR2 polypeptide. One such nucleobase oligomer, is referred to as a Peptide Nucleic Acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an

aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Methods for making and using these nucleobase oligomers are described, for example, in "Peptide Nucleic Acids: Protocols and Applications" Ed. P. E. Nielsen, Horizon Press, Norfolk, United Kingdom, 1999. Representative United States patents that teach the preparation of PNAs include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et ak, Science, 1991, 254, 1497-1500.

In particular embodiments of the invention, the nucleobase oligomers have phosphorothioate backbones and nucleosides with heteroatom backbones, and in particular - CH_2-NH-0-CH₂-, -CH₂-N(CH₃)-0-CH₂- (known as a methylene (methylimino) or MMI backbone), -CH₂-0-N(CH )-CH₂-, -CH₂-N(CH )-N(CH )-CH₂-, and -0-N(CH )-CH₂-CH₂-.

In other embodiments, the oligonucleotides have morpholino backbone structures described in U.S. Pat. No. 5,034,506.

Nucleobase oligomers may also contain one or more substituted sugar moieties.

Nucleobase oligomers comprise one of the following at the 2' position: OH; F; 0-, S-, or N- alkyl; 0-, S-, or N-alkenyl; 0-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C₂ to Cio alkenyl and alkynyl. Particularly preferred are 0[(CH₂)_n0] _nCH₃, 0(CH₂) _nOCH₃, 0(CH₂) _nNH₂, 0(CH₂) _nCH₃, 0(CH₂) _nONH₂, and 0(CH₂) nON[(CH₂) _nCH₃)]₂, where n and m are from 1 to about 10. Other preferred nucleobase oligomers include one of the following at the 2' position: Ci to Cio lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O- aralkyl, SH, SCH , OCN, Cl, Br, CN, CF , OCF , SOCH , S0₂CH , 0N0₂, N0₂, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkyl amino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the

pharmacokinetic properties of a nucleobase oligomer, or a group for improving the pharmacodynamic properties of an nucleobase oligomer, and other substituents having similar properties. Preferred modifications are 2'-0-methyl and 2'-methoxyethoxy (2'-0- CH2CH2OCH3, also known as 2'-0-(2-methoxyethyl) or 2'-MOE). Another desirable modification is 2'-dimethylaminooxyethoxy (i.e., O(CFh) ₂0N(CH₃) 2), also known as 2'- DMAOE. Other modifications include, 2'-aminopropoxy ^'-OCFhCFhCFhNFh) and 2'- fluoro (2'-F). Similar modifications may also be made at other positions on an

oligonucleotide or other nucleobase oligomer, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Nucleobase oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative ETnited States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference in its entirety.

Nucleobase oligomers may also include nucleobase modifications or substitutions. As used herein, "unmodified" or "natural" nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me- C), 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine; 5-halouracil and cytosine; 5- propynyl uracil and cytosine; 6-azo uracil, cytosine and thymine; 5 -uracil (pseudouracil); 4- thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7- deazaguanine and 7-deazaadenine; and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in ET.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. F, ed. John Wiley & Sons, 1990, those disclosed by Englisch et ak, Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of an antisense oligonucleotide of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and 0-6 substituted purines, including 2- aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine

substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2. degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are desirable base substitutions, even more particularly when combined with 2'-0-methoxyethyl or 2'-0-methyl sugar modifications. Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include U.S. Pat. Nos.

4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and 5,750,692, each of which is herein incorporated by reference.

Another modification of a nucleobase oligomer of the invention involves chemically linking to the nucleobase oligomer one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et ah, Proc. Natl. Acad. Sci. USA, 86:6553-6556, 1989), cholic acid (Manoharan et ah, Bioorg. Med. Chem. Let, 4: 1053-1060, 1994), a thioether, e.g., hexyl -S-trityl thiol (Manoharan et ah, Ann. N.Y. Acad. Sci., 660:306-309, 1992; Manoharan et ah, Bioorg. Med. Chem. Let., 3:2765-2770, 1993), a thiocholesterol (Oberhauser et ah, Nucl. Acids Res., 20:533-538: 1992), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et ah, EMBO L, 10:1111- 1118, 1991; Kabanov et ah, FEBS Lett., 259:327-330, 1990; Svinarchuk et ah, Biochimie, 75:49-54, 1993), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium l,2-di- O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et ah, Tetrahedron Lett., 36:3651- 3654, 1995; Shea et ah, Nucl. Acids Res., 18:3777-3783, 1990), a polyamine or a

polyethylene glycol chain (Manoharan et ah, Nucleosides & Nucleotides, 14:969-973, 1995), or adamantane acetic acid (Manoharan et ah, Tetrahedron Lett., 36:3651-3654, 1995), a palmityl moiety (Mishra et ah, Biochim. Biophys. Acta, 1264:229-237, 1995), or an octadecyl amine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et ah, J. Pharmacol. Exp. Then, 277:923-937, 1996. Representative United States patents that teach the preparation of such nucleobase oligomer conjugates include U.S. Pat. Nos. 4,587,044;

4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,828,979; 4,835,263; 4,876,335; 4,904,582; 4,948,882; 4,958,013; 5,082,830; 5,109,124; 5,112,963; 5,118,802; 5,138,045; 5,214,136; 5,218,105; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,414,077; 5,416,203, 5,451,463; 5,486,603; 5,510,475; 5,512,439; 5,512,667; 5,514,785; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,565,552; 5,567,810; 5,574,142; 5,578,717; 5,578,718; 5,580,731; 5,585,481; 5,587,371; 5,591,584; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,608,046; and 5,688,941, each of which is herein incorporated by reference.

The present invention also includes nucleobase oligomers that are chimeric compounds. "Chimeric" nucleobase oligomers are nucleobase oligomers, particularly oligonucleotides, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide. These nucleobase oligomers typically contain at least one region where the nucleobase oligomer is modified to confer, upon the nucleobase oligomer, increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the nucleobase oligomer may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of nucleobase oligomer inhibition of gene expression. Consequently, comparable results can often be obtained with shorter nucleobase oligomers when chimeric nucleobase oligomers are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region.

Chimeric nucleobase oligomers of the invention may be formed as composite structures of two or more nucleobase oligomers as described above. Such nucleobase oligomers, when oligonucleotides, have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.

The nucleobase oligomers used in accordance with this invention may be

conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. The nucleobase oligomers of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative ETnited States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543, 158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5, 108,921; 5,213,804; 5,227, 170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.

ACC Therapy

The therapeutic methods of the invention in general comprise administration of a therapeutically effective amount of the agents described herein, such as, for example a small molecule inhibitor of Fgfr2 or an inhibitory polynucleotide (e.g., siRNA, shRNA, antisense oligonucleotide) to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like).

Inhibitors of Fgfr2 activity are known in the art and include small molecule inhibitors and monoclonal antibodies specific for Fgfr2. Inhibitors of Fgfr2 activity include, without limitation, AZD4547, a tyrosine kinase inhibitor which targets FGFR1-3 (available from AstraZeneca); BGJ398 (infigratinib), a Pan-FGF receptor kinase inhibitor (available from Novartis); Bemarituzumab (FPA144), a monoclonal antibody that binds to FGFR2b preventing binding of certain FGFs (available from Five Prime); Alofanib (RPT835), a novel first-in-class allosteric small-molecular inhibitor of FGFR2 (available from Ruspharmtech); and SSR128129E, an allosteric inhibitor of FGF receptor signaling (available from Sanofi Aventis).

The compositions and methods of the invention can be used alone or in combination with conventional therapies for ACC, which include tumor resection, radiation, mitotane adjuvant therapy, and one or more of the following Streptozotocin plus mitotane, Etoposide, doxorubicin, and cisplatin plus mitotane. In cases of increased hormone production, anti steroidogenic drugs such as ketoconazole and metyrapone, and steroid receptor antagonists, such as spironolactone and mifepristone, should be considered.

Pharmaceutical Compositions

For therapeutic use, a compound or agent (e.g., small molecule inhibitor of Fgfr2) or a pharmaceutically acceptable salt thereof is formulated with a carrier that is pharmaceutically acceptable and is appropriate for delivering the compound or agent by the chosen route of administration. Suitable pharmaceutically acceptable carriers are those used conventionally with small molecules, such as diluents, excipients and the like. See, for example, "Remington s Pharmaceutical Sciences", l7th Ed., Mack Publishing Company, Easton, Pa., 1995, for guidance on drug formulations. In one embodiment, the compounds are formulated for administration by infusion or by injection, either subcutaneously or intravenously, and are accordingly utilized as aqueous solutions in sterile and pyrogen-free form and optionally buffered to a slightly acidic or physiological pH. Thus, the compounds/agents may be administered in distilled water, saline, buffered saline or 5% dextrose solution. Water solubility of compositions comprising a compound or agent may be enhanced by

incorporating a solubility enhancer, such as acetic acid.

Methods of Delivery

Compounds/agents and compositions comprising same may be administered via a variety of methods. Such methods include, without limitation, intravesicular, intralesional (in and around an adrenal gland), oral, intravenous (iv), subcutaneous (sc or sq), intraperitoneal, intramuscular intradermal, rectal, nasal, or topical administration, or inhalation via nebulizer or inhaler, to a subject (e.g., a mammal) in need thereof.

Therapeutic Dosing and Regimen

The therapeutic dosing and regimen best suited for treatment of a subject (e.g., a human patient) vary with the disorder or condition to be treated, and according to the patient's weight and other parameters. A dose of at least one compound/agent described herein may, for example, be administered at about 2.5 mg/kg, administered twice daily over 10 days. Smaller doses, e.g., in the pg/kg range, and shorter or longer duration or frequency of treatment, are also envisioned to produce therapeutically useful results, i.e., a statistically significant decrease in cell proliferation and/or cancer. It is, moreover, envisioned that localized administration to, e.g., at least one adrenal gland, may be optimized based on the response of adrenal cells therein.

An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject, including the size, age, and general condition of the patient, the particular disorder being treated, the severity of the disorder, and the presence of other drugs in the patient. Trial dosages may be chosen after consideration of the results of animal studies and the clinical literature.

A typical human dose of a compound/agent may be from about 10 pg/kg body weight/day to about 10 mg/kg/day, more particularly from about 50 pg/kg/day to about 5 mg/kg/day, and even more particularly about 100 pg/kg/day to 1 mg/kg/day.

Therapeutic efficacy of a compound/agent and/or compositions comprising same may be determined by evaluating and comparing patient symptoms and quality of life pre- and post-administration. Such methods apply irrespective of the mode of administration. In a particular embodiment, pre-administration refers to evaluating patient symptoms and quality of life prior to onset of therapy and post-administration refers to evaluating patient symptoms and quality of life at least 2-8 weeks after onset of therapy. In a particular embodiment, the post-administration evaluating is performed about 2-8, 2-6, 4-6, or 4 weeks after onset of therapy. In a particular embodiment, patient symptoms (e.g., cancer) and quality of life pre- and post-administration are evaluated via questionnaire assessment.

In some embodiments, the formulation comprising a compound/agent comprises one or more additional components, wherein the additional component is at least one of an osmolar component that provides an isotonic, or near isotonic solution compatible with human cells or blood, and a preservative.

In some embodiments, the osmolar component is a salt, such as sodium chloride, or a sugar or a combination of two or more of these components. In some embodiments, the sugar may be a monosaccharide such as dextrose, a disaccharide such as sucrose or lactose, a polysaccharide such as dextran 40, dextran 60, or starch, or a sugar alcohol such as mannitol. The osmolar component is readily selected by those skilled in the art.

In some embodiments, the preservative is at least one of parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In some embodiments, the formulation comprising a compound/agent is in the form of a sustained release formulation and further comprises one or more additional components, wherein the additional component is at least one of an anti-inflammatory agent; and a preservative.

In some embodiments, the sustained release formulation is administered as a suppository.

In some embodiments, the sustained release formulation is administered in an implant designed for subcutaneous (sc or sq) implantation. Exemplary sc implants are known to those of skill in the art and may involve a port or catheter or the like. In a particular embodiment, the port or catheter is implanted in or near an adrenal gland.

The practice of the present invention employs, unless otherwise indicated,

conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as,

“Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989);

“Oligonucleotide Synthesis” (Gait, 1984);“Animal Cell Culture” (Freshney, 1987);

“Methods in Enzymology”“Handbook of Experimental Immunology” (Weir, 1996);“Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987);“Current Protocols in Molecular Biology” (Ausubel, 1987);“PCR: The Polymerase Chain Reaction”, (Mullis, 1994);“Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1: Stabilization o b-catenin in zG cells results in ectopic accumulation of the zG.

Dysregulation of WNT/p-catenin signaling and TP 53 mutations have been implicated in both sporadic and familial forms of adrenocortical carcinoma (ACC). In fact, genomic characterization has revealed that these pathways are the most frequently altered in ACCs with the poorest outcomes. Despite their critical role in the development and prognosis of ACC, it remains unclear whether the combination of Trp53 loss and b-catenin gain-of- function mutations can efficiently initiate a neoplastic process. To investigate this, the Cypllb2(AS)^Cre mouse model was utilized to generate transgenic mice with adrenal-specific (1) Trp53 deletion ( Trp53^Floxed ), (2) b-catenin gain-of-function (Ctnnb l^FloxedEx3) (FIG. 1), or (3) Trp53 deletion and b-catenin gain-of-function (Trp53^Floxed::Ctnnbl^FloxedEx3).

Analysis of mice with targeted deletion of Trp53 at 1, 3 and 5 months of age showed no gross morphological or histological changes compared with controls as evidenced by normal b-catenin localization at the Zona glomerulosa with a well-organized zona fasciculate. These adrenals had an increased weight but a normal ki67 count. Gene expression analysis of whole adrenals showed decreased levels of p2J Axin2 and Shh at 5 months of age. These findings indicate that Trp53 loss leads to a decrease in the p2l tumor suppressor, as well as a down-regulation of canonical Wnt signaling and progenitor cell activity. These results suggest that a secondary event is required to override the suppression of Wnt signaling, a key feature of ACC, to promote neoplasia

zG-specific stabilization of b-catenin ( Cypllb2(AS)^Cre/⁺ :: Ctnnbl^FIoxedEx3) ^cat-GOF] mice have a conditional deletion of Ctnnbl exon 3 following Cre-mediated recombination that results in stabilization of b-catenin and constitutive activation of the canonical (c]WNT pathway] specifically within zG cells.

Floxed p53 mice were obtained that include an intact wildtype p53 allele containing two loxP recombination sites (p53F/F) in introns 1 and 10 of p53. When these p53flox mice are bred to mice with a Cre recombinase gene under the control of a promoter of interest, Trp53 expression is deleted in the tissue of interest. These Trp53 knock-out mice were used to obtain mice having zona granulosa (zG)-specific beta catenin gain-of-function (GOF) and p53 loss-of-function (LOF).

Analysis of mice with targeted b-catenin gain-of-function at 1 and 3 months of age showed an expansion of the zona glomerulosa (zG), at the expense of the zona fasciculata (zF), without an overall increase in adrenal size compared with controls. Gene expression analysis showed increased expression of Axin2 and Shh , as well as the ACC-related genes Ezh2 and IGF2. These results suggest that activation of Wnt signaling alone is not sufficient to drive neoplastic transformation.

In bq8ΐ^T adrenals b-catenin expression was restricted to a thin layer of cells beneath the capsule at 1 and 3 months (Figure 2A and 2B] In contrast, boaI-ΰOR adrenals showed progressive expansion of b-catenin expressing cells extending into the orthotopic zF (Figure 2G and 2H). Trp53 KO mice having a loss of function in p53 had b-catenin expression in a thin layer of cells beneath the capsule at 1 and 3 months (FIG. 2D, E), just as wild-type mice did.

Interestingly, mice having both a b-catenin GOF and a p53 LOF (b-catenin GOF/p53 LOF ) showed adenoma formation (FIG. 21, J).

Tumor formation in b-catenin GOF/p53 LOF mice was fully penetrant (FIG. 3). Wild- type and p53 LOF mice remained tumor free throughout the course of the experiment (>50 weeks]. In contrast, mice having both a b-catenin GOF and a p53 LOF all developed tumors at about 30+ weeks of age.

Analysis of mice with the combined Trp53 and b-catenin mutations showed a dramatic expansion of the zG, compared with b-catenin gain-of-function mice, at 1 month of age, and enlarged, hyperplastic adrenals, with focal nodularity at 3 months of age. Gene expression analysis showed elevated expression of Axin2 , Shh and Ezh2, but low levels of p21. Changes in gene expression of Ezh2 in mice having b-catenin gain-of-function (Kcat- GOF] or Trp53 deletion and b-catenin gain-of-function (Trp53 KO Kcat-GOF] are shown at F1G. 7. These results indicate that the combination of Trp53 deletion and b-catenin gain- of-function is sufficient to initiate neoplastic changes in the adrenal cortex.

At 38 weeks, some of these mice developed Adrenocortical carcinoma (ACC) with metastasis (FIG. 3).

Hormonal activity in the b-catenin GOF/p53 LOF mice was also disrupted (FIG. 4). Increased levels of aldosterone, corticosterone, ACTH, and in the corticosterone/ACTH ratio were observed. These changes were consistent with hyperaldosteronism and Cushing’s Syndrome.

FGFR2 isoforms were upregulated in the b-catenin GOF/p53 LOF mice (FIG. 5). Loss of FGFR2 impacts zG identity (FIG. 8). Inhibitors of FGFR2 (AZD4547 and BGJ398) were used to assess the effect of FGFR2 inhibition on zG proliferation and size (FIG. 6).

To determine the impact FGFR inhibitors have on zG function. Mice were treated with 30 mg/kg/day per os (p.o.) of AZD4547 (N=6) or with vehicle only (N=6) starting on day 4. Aldosterone levels for the mice receiving the drug and mice receiving vehicle only were compared using two-way ANOVA analysis followed by Bonferroni’s multiple comparison correction test. Decreased levels of aldosterone were observed in treated mice compared to untreated mice (FIG. 9A). An additional cohort of mice were treated with 30 mg/kg/day p. o. of BGJ398 for 7 days. qRT-PCR analysis was performed to determine Cypl lb2 and Cyclin Dl transcript levels in adrenals from treated and control mice. The results were analyzed using two sample t-test. Both Cypl lb2 and Cyclin Dl transcript levels were reduced in treated mice compared to untreated controls (FIG. 9B). Taken together, these results indicate that FGFR inhibitors impair zG function.

The impact of Fgfr2 deletion on zG hyperplasia driven by b-catenin gain-of-function was determined. Adult mouse adrenals were isolated and imaged for GFP, DAPI, and Dab2 co-immunostaining. The adult mice belonged to one of the following cohorts: control (ASCre/+; mTmG), PC at GOF (ASCre/+; mTmG; b-catenin fl(ex3)/+), and pCat GOF; Fgfr2 cKO (ASCre/+; mTmG; b-catenin fl(ex3)/+; Fgfr2 fl/fl). Referring to FIG. 10, the representative images demonstrate that Fgfr2 deletion prevents zG hyperplasia driven by b- catenin gain-of-function.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A method of inhibiting cell proliferation in Adrenocortical Carcinoma, the method comprising contacting a cell of the Adrenocortical Carcinoma with an agent that inhibits Fibroblast Growth Factor Receptor 2 (FGFR2) activity.

2. A method for treating Adrenocortical Carcinoma in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent that inhibits Fibroblast Growth Factor Receptor 2 (FGFR2) activity.

3. The method of claim 1 or 2, wherein agent is a small molecule, protein, or polynucleotide.

4. The method of claim 3, wherein the agent binds to FGFR2 and inhibits ligand binding.

5. The method of claim 3, wherein the agent is a tyrosine kinase inhibitor.

6. The method of claim 3, wherein the agent is a small molecule inhibitor, an antibody specific for fgfr2, or an antigen-binding fragment of an antibody specific for FGFR2.

7. The method of claim 3, wherein the small molecule inhibitor is selected from the group consisting of AZD4547, BGJ398 (infigratinib), Alofanib (RPT835), and SSR128129E; and the protein is Bemarituzumab (FPA144).

8. The method of claim 3, wherein the agent inhibits FGFR2 expression.

9. The method of claim 3, wherein the agent is a polynucleotide.

10. The method of claim 6, wherein the agent is an inhibitory nucleic acid molecule.

11. The method of claim 10, wherein the inhibitory nucleic acid molecule is an antisense nucleic acid molecule, siRNA or a vector encoding an inhibitory nucleic acid molecule.

12. The method of claim 2, wherein the subject is a mammal.

13. The method of claim 12, wherein the mammal is a human.

14. The method of claim 2, wherein the composition is administered intravenously, subcutaneously, intraperitoneally, orally, via inhalation, or locally.

15. The method of claim 14, wherein the administering involves direct administration into and around the adrenal gland.