WO2015048367A1 - Fusion proteins and methods of use thereof - Google Patents

Abstract

The invention discloses oncogenic fusion proteins. The invention provides methods for treating gene-fusion associated cancers.

Description

FUSION PROTEINS AND METHODS OF USE THEREOF

[0001] This application claims priority to U.S. Provisional Application No. 61/882,929, filed September 26, 2013, the contents of which are hereby incorporated by reference in their entirety.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

BACKGROUND OF THE INVENTION

[0004] Childhood cancer of the liver is a rare disease in which malignant cells are found in the liver. It is divided into two major histologic subgroups: hepatoblastoma and hepatocellular carcinoma. The incidence of hepatic tumors in children 14 years and younger is 2.4 per 100,000, of which 2 per 100,000 are hepatoblastoma.

[0005] Fibrolamellar Hepatocellular Carcinoma (FHC) is a rare cancer of the liver, which typically affects yound adults. The age-adjusted incidence rate of FHC in the United States is 0.02 per 100,000 (El-Serag et al. (2004) Hepatology, 39(3):798-803).

SUMMARY OF THE INVENTION

[0006] The invention is based, at least in part, on the discovery of highly expressed gene fusions in pediatric liver cancer (e.g., Fibrolamellar Hepatocellular Carcinoma (FHC)), which join the J domain of heat shock protein genes (e.g., DNAJB1) to the kinase domain of a cAMP- dependent protein kinase (e.g., PRKACA). The invention is based, at least in part, on the finding that DNA J-cAMP-dependent PK fusion molecules identify a subset of liver cancer patients who will benefit from targeted inhibition of the kinase activity of a cAMP-dependent protein kinase. Identification of DNA J-cAMP-dependent PK fusions in liver cancer patients are useful therapeutic targets, as well as for the development of diagnostic tests.

[0007] An aspect of the invention provides for a purified fusion protein comprising a heat shock protein fused to a kinase domain of a cAMP-dependent protein kinase. [0008] An aspect of the invention provides for a purified fusion protein comprising a J domain fused to a kinase domain of a cAMP-dependent protein kinase. In one embodiment, the heat shock protein is a DNA J protein. In another embodiment, the DNA J protein is a DNAJB 1 protein. In a further embodiment, the cAMP-dependent protein kinase is cAMP-dependent protein kinase catalytic subunit alpha protein (PRKACA).

[0009] An aspect of the invention provides for a purified fusion protein comprising a DNA J protein fused to a kinase domain of a cAMP-dependent protein kinase. In one embodiment, the DNA J protein is a DNAJB 1 protein. In a further embodiment, the cAMP-dependent protein kinase is cAMP-dependent protein kinase catalytic subunit alpha protein (PRKACA).

[0010] An aspect of the invention provides for a purified fusion protein comprising a DNAJB 1 protein fused to a kinase domain of a cAMP-dependent protein kinase. In a further embodiment, the cAMP-dependent protein kinase is cAMP-dependent protein kinase catalytic subunit alpha protein (PRKACA).

[0011] An aspect of the invention provides for a purified fusion protein comprising a DNAJB 1 protein fused to a cAMP-dependent protein kinase catalytic subunit alpha protein (PRKACA).

[0012] An aspect of the invention provides for a purified fusion protein encoded by an

DNAJB 1 -PRKACA nucleic acid, wherein DNAJB 1 -PRKACA comprises exon 1 of DNAJB 1 located on human chromosome 19 spliced 5' to a combination of exons 2-10 of PRKACA located on human chromosome 19.

[0013] An aspect of the invention provides for a purified DNAJB 1 -PRKACA fusion protein comprising SEQ ID NO: 1.

[0014] An aspect of the invention provides for a synthetic nucleic acid encoding the fusion proteins described herein.

[0015] An aspect of the invention provides for a synthetic nucleic acid encoding a DNAJB 1- PRKACA fusion protein, wherein the nucleic acid comprises SEQ ID NO: 2.

[0016] An aspect of the invention provides for an antibody or antigen-binding fragment thereof, that specifically binds to a purified fusion protein comprising a DNA J protein fused to a kinase domain of a cAMP-dependent protein kinase. In one embodiment, the DNA J protein is a DNAJB 1 protein. In another embodiment, the cAMP-dependent protein kinase is PRKACA. In a further embodiment, the fusion protein is a DNAJB 1 -PRKACA fusion protein. In some embodiments, the DNAJBl-PRKACA fusion protein comprises the amino acid sequence of SEQ ID NO: 1.

[0017] An aspect of the invention provides for a composition for decreasing in a subject the expression level or activity of a fusion protein comprising a DNA J protein fused to a kinase domain of a cAMP-dependent protein kinase, the composition in an admixture of a

pharmaceutically acceptable carrier comprising an inhibitor of the fusion protein. In one embodiment, the fusion protein is DNAJBl-PRKACA fusion protein. In another embodiment, the inhibitor comprises an antibody that specifically binds to a DNAJBl-PRKACA fusion protein or a fragment thereof; a small molecule that specifically binds to a DNAJB1 protein; a small molecule that specifically binds to a PRKACA protein; an antisense RNA or antisense DNA that decreases expression of a DNAJBl-PRKACA fusion polypeptide; a siRNA that specifically targets a DNAJBl-PRKACA fusion gene; or a combination thereof. In a further embodiment, the small molecule that specifically binds to a PRKACA protein comprises 5-Iodotubercidin, A-3

Hydrochloride, KT 5720, ML-9, H-89, HA-1004, H7, A-674563, K252, or a combination thereof.

[0018] An aspect of the invention provides for a method for decreasing in a subject in need thereof the expression level or activity of a fusion protein comprising a DNA J protein fused to a kinase domain of a cAMP-dependent protein kinase. In one embodiment, the method comprises administering to the subject a therapeutic amount of a composition for decreasing in a subject the expression level or activity of a fusion protein comprising a DNA J protein fused to a kinase domain of a cAMP-dependent protein kinase, the composition in an admixture of a

pharmaceutically acceptable carrier comprising an inhibitor of the fusion protein; and determining whether the fusion protein expression level or activity is decreased compared to fusion protein expression level or activity prior to administration of the composition, thereby decreasing the expression level or activity of the fusion protein.

[0019] An aspect of the invention provides for a method for treating a liver cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a DNA J-cAMP-dependent PK fusion molecule inhibitor. In one embodiment, the liver cancer comprises fibrolamellar hepatocellular carcinoma, hepatoblastoma, hepatocellular carcinoma, hepatocellular adenoma, cavernous hemangioma, or a combination of the liver cancers described. In another embodiment, the DNA J-cAMP-dependent PK fusion molecule inhibitor specifically binds to a DNAJBl-PRKACA fusion protein or a fragment thereof. In a further embodiment, the inhibitor comprises an antibody that specifically binds to a DNAJBl-PRKACA fusion protein or a fragment thereof; a small molecule that specifically binds to a DNAJB 1 protein; a small molecule that specifically binds to a PRKACA protein; an antisense RNA or antisense DNA that decreases expression of a DNAJBl-PRKACA fusion polypeptide; a siRNA that specifically targets a

DNAJBl-PRKACA fusion gene; or a combination of the inhibitors described. In some

embodiments, the small molecule that specifically binds to a PRKACA protein comprises 5- Iodotubercidin, A-3 Hydrochloride, KT 5720, ML-9, H-89, HA-1004, H7, A-674563, K252, or a combination of the molecules described.

[0020] An aspect of the invention provides for a method of decreasing growth of a solid tumor in a liver of a subject in need thereof, the method comprising administering to the subject an effective amount of a DNA J-cAMP-dependent PK fusion molecule inhibitor, wherein the inhibitor decreases the size of the solid tumor. In one embodiment, the solid tumor comprises fibrolamellar hepatocellular carcinoma, hepatoblastoma, hepatocellular carcinoma, hepatocellular adenoma, cavernous hemangioma, or a combination of the tumors described. In another embodiment, the DNA J-cAMP-dependent PK fusion molecule inhibitor specifically binds to a DNAJBl-PRKACA fusion protein or a fragment thereof. In a further embodiment, the inhibitor comprises an antibody that specifically binds to a DNAJBl-PRKACA fusion protein or a fragment thereof; a small molecule that specifically binds to a DNAJB 1 protein; a small molecule that specifically binds to a PRKACA protein; an antisense RNA or antisense DNA that decreases expression of a DNAJBl- PRKACA fusion polypeptide; a siRNA that specifically targets a DNAJBl-PRKACA fusion gene; or a combination of the inhibitors described. In some embodiments, the small molecule that specifically binds to a PRKACA protein comprises 5-Iodotubercidin, A-3 Hydrochloride, KT 5720, ML-9, H-89, HA-1004, H7, A-674563, K252, or a combination of the molecules described.

[0021] An aspect of the invention provides for a diagnostic kit for determining whether a sample from a subject exhibits a presence of a DNAJBl-PRKACA fusion molecule. In one embodiment, the kit comprises at least one oligonucleotide that specifically hybridizes to the DNAJBl-PRKACA fusion molecule, or a portion thereof. In one embodiment, the

oligonucleotides comprise a set of nucleic acid primers or in situ hybridization probes. In another embodiment, the oligonucleotide recognizes a nucleic acid comprising SEQ ID NO: 2. In a further embodiment, the primers prime a polymerase reaction only when a DNAJBl-PRKACA fusion is present. In some embodiments, the determining comprises gene sequencing, selective

hybridization, selective amplification, gene expression analysis, or a combination thereof.

[0022] An aspect of the invention provides for a diagnostic kit for determining whether a sample from a subject exhibits a presence of a DNAJBl-PRKACA fusion protein. In one embodiment, the kit comprising an antibody that specifically binds to a DNAJBl-PRKACA fusion protein comprising SEQ ID NO: 1, wherein the antibody will recognize the protein only when a DNAJBl-PRKACA fusion protein is present.

[0023] An aspect of the invention provides for a method for detecting the presence of a

DNAJBl-PRKACA fusion in a human subject. In one embodiment, the method comprises obtaining a biological sample from the human subject; and detecting whether or not there is a DNAJBl-PRKACA fusion present in the subject. In one embodiment, the detecting comprises measuring DNAJBl-PRKACA fusion protein levels by ELISA using an antibody directed to SEQ ID NO: 1; western blot using an antibody directed to SEQ ID NO: 1; mass spectroscopy, isoelectric focusing, or a combination thereof.

[0024] An aspect of the invention provides for a method for detecting the presence of a

DNAJBl-PRKACA fusion in a human subject. In one embodiment, the method comprises obtaining a biological sample from a human subject; and detecting whether or not there is a nucleic acid sequence encoding a DNAJBl-PRKACA fusion protein in the subject. In another

embodiment, the nucleic acid sequence comprises SEQ ID NO: 2. In a further embodiment, the detecting comprises using hybridization, amplification, or sequencing techniques to detect a DNAJBl-PRKACA fusion. In some embodiments, the amplification uses primers directed to SEQ ID NO: 2.

BRIEF DESCRIPTION OF THE FIGURES

[0025] FIG. 1 is a ribbon diagram of the complex between the catalytic and regulatory (RIa) subunits of PKA. This fusion truncates the N-terminal 18 amino acids of the protein kinase catalytic subunit.

DETAILED DESCRIPTION OF THE INVENTION

[0026] Childhood cancer of the liver is a rare disease in which malignant cells are found in the liver. There are two types of primary liver cancer: hepatoblastoma and hepatocellular cancer, which are based on the histology of cancer cells. Hepatoblastoma is more common in young children before the age of 3 and may be caused by abnormal gene expression. Approximately 90% of malignant liver tumors in children aged 4 years and younger are hepatoblastomas. The overall survival rate for children with hepatoblastoma is 70%, but is only 25% for those with hepatocellular carcinoma. See a review of pediatric tumors: Emre et al, (2012) Pediatr Transplant. 16(6):549-63, which is incorporated by reference in its entirety. [0027] Fibrolamellar Hepatocellular Carcinoma (FHC) is a rare cancer of the liver. It typically affects young adults and is histologically characterized by laminated fibrous layers interspersed between the tumor cells. This form of cancer is often advanced when diagnosed due to lack of symtpoms. FHC does not produce the alpha fetoprotein biomarker, typically observed in hepatocellular carcinoma. However, elevated neurotensin levels have been observed in FHC patients. See reviews of FHC: Mavros et al. (2012) J Am Coll Surg. 215(6):820-30; Chun et al, (2013) Recent Results Cancer Res. 190: 101-10; and Paradis (2013) Recent Results Cancer Res. 190:21-32, each of which are hereby incorporated by reference in their entireties.

[0028] Chaperone DnaJ, also known as Heat Shock p40 (Hsp40, 40 kD), is a molecular chaperone protein. It protects proteins from aggregation during synthesis and during cellular stress. It consists of three domains: the N-terminal domain comprising the J domain; a central domain comprising a cysteine rich region (zinc-finger domain); and the C-terminal domain which functions in dimerization and chaperoning. Non-limiting examples of proteins containing a J domain include: DNAJA1; DNAJA2; DNAJ A3; DNAJA4; DNAJB1; DNAJB11; DNAJB13; DNAJB4; DNAJB5; MST104. See a review of Chaperone DnaJ proteins: Kakkar et al., (2012) Curr Top Med

Chem.12(22):2479-90), which is incorporated by reference in its entirety.

[0029] A cAMP-dependent protein kinase comprises a family of protein kinases, and is also known as protein kinase A (PKA). It is an enzyme whose activity is dependent on cellular levels of cyclic AMP (cAMP). cAMP-dependent protein kinase catalytic subunit alpha (PRKACA), a member of the family, is an enzyme that in humans is encoded by the PRKACA gene. See for example, Taylor et al, (2013) Biochim Biophys Acta. 1834(7): 1271-8), which is incorporated by reference in its entirety.

DNA and AminoAcid Manipulation Methods and Purification Thereof

[0030] The practice of aspects of the present invention can employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Molecular Cloning A Laboratory Manual, 3^rd Ed., ed. by Sambrook (2001), Fritsch and Maniatis (Cold Spring Harbor Laboratory Press:

1989); DNA Cloning, Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M. J. Gait ed., 1984); MuUis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In Enzymology (Academic Press, Inc., N.Y.), specifically, Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Immunochemical Methods In Cell And

Molecular Biology (Caner and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M. Weir and C. C. Blackwell, eds., 1986);

Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986). All patents, patent applications and references cited herein are incorporated by reference in their entireties.

[0031] One skilled in the art can obtain a protein in several ways, which include, but are not limited to, isolating the protein via biochemical means or expressing a nucleotide sequence encoding the protein of interest by genetic engineering methods.

[0032] A protein is encoded by a nucleic acid (including, for example, genomic DNA, complementary DNA (cDNA), synthetic DNA, as well as any form of corresponding RNA). For example, it can be encoded by a recombinant nucleic acid of a gene. The proteins of the invention can be obtained from various sources and can be produced according to various techniques known in the art. For example, a nucleic acid that encodes a protein can be obtained by screening DNA libraries, or by amplification from a natural source. A protein can be a fragment or portion thereof. The nucleic acids encoding a protein can be produced via recombinant DNA technology and such recombinant nucleic acids can be prepared by conventional techniques, including chemical synthesis, genetic engineering, enzymatic techniques, or a combination thereof. For example, a fusion protein of the invention comprises the J domain of a heat shock protein fused to a kinase domain of a cAMP-dependent protein kinase. In one embodiment, a fusion protein of the invention comprises a DNA J protein fused to a kinase domain of a cAMP-dependent protein kinase. In another embodiment, a fusion protein of the invention comprises a DNAJB1 protein fused to a kinase domain of a cAMP-dependent protein kinase. In a further embodiment, a fusion protein of the invention comprises a DNAJB1 protein fused to a cAMP-dependent protein kinase catalytic subunit alpha protein. In some embodiments, the cAMP-dependent protein kinase is cAMP- dependent protein kinase catalytic subunit alpha protein (PRKACA). In some embodiments, the fusion protein is a DNAJB1 -PRKACA fusion protein. An example of a DNAJB1 -PRKACA polypeptide has the amino acid sequence shown in SEQ ID NO: 1.

[0033] The Genbank ID for the PRKACA gene is 5566. Two isoforms are listed for PRKACA, e.g., having Genebank Accession Nos. NP_002721 (corresponding nucleotide sequence NM_002730); and NP_997401 (corresponding nucleotide sequence NM_207518).

[0034] SEQ ID NO: 3 is the PRKACA Amino Acid Sequence, Transcript Variant 1 (NP_002721; 351 aa).

1 MGNAAAAKKG SEQESVKEFL AKAKEDFLKK WESPAQNTAH LDQFERIKTL GTGSFGRVML 61 VKHKETGNHY AMKILDKQKV VKLKQIEHTL NEKRILQAVN FPFLVKLEFS FKDNSNLYMV 121 MEYVPGGEMF SHLRRIGRFS EPHARFYAAQ IVLTFEYLHS LDLIYRDLKP ENLLIDQQGY 181 IQVTDFGFAK RVKGRTWTLC GTPEYLAPEI ILSKGYNKAV DWWALGVLIY EMAAGYPPFF 241 ADQPIQIYEK IVSGKVRFPS HFSSDLKDLL RNLLQVDLTK RFGNLKNGVN DIKNHKWFAT 301 TDWIAIYQRK VEAPFIPKFK GPGDTSNFDD YEEEEIRVSI NEKCGKEFSE F

[0035] SEQ ID NO: 4 is the PRKACA Nucleotide Sequence, Transcript Variant 1 (NM_002730; 2689 bp).

1 gatcttgggc tgaggttccc gggcgggcgg gcgcggagag acgcgggaag caggggctgg 61 gcgggggtcg cggcgccgca gctagcgcag ccagcccgag ggccgccgcc gccgccgccc 121 agcgcgctcc ggggccgccg gccgcagcca gcacccgccg cgccgcagct ccgggaccgg 181 ccccggccgc cgccgccgcg atgggcaacg ccgccgccgc caagaagggc agcgagcagg 241 agagcgtgaa agaattctta gccaaagcca aagaagattt tcttaaaaaa tgggaaagtc 301 ccgctcagaa cacagcccac ttggatcagt ttgaacgaat caagaccctc ggcacgggct 361 ccttcgggcg ggtgatgctg gtgaaacaca aggagaccgg gaaccactat gccatgaaga 421 tcctcgacaa acagaaggtg gtgaaactga aacagatcga acacaccctg aatgaaaagc 481 gcatcctgca agctgtcaac tttccgttcc tcgtcaaact cgagttctcc ttcaaggaca 541 actcaaactt atacatggtc atggagtacg tgcccggcgg ggagatgttc tcacacctac 601 ggcggatcgg aaggttcagt gagccccatg cccgtttcta cgcggcccag atcgtcctga 661 cctttgagta tctgcactcg ctggatctca tctacaggga cctgaagccg gagaatctgc 721 tcattgacca gcagggctac attcaggtga cagacttcgg tttcgccaag cgcgtgaagg 781 gccgcacttg gaccttgtgc ggcacccctg agtacctggc ccctgagatt atcctgagca 841 aaggctacaa caaggccgtg gactggtggg ccctgggggt tcttatctat gaaatggccg 901 ctggctaccc gcccttcttc gcagaccagc ccatccagat ctatgagaag atcgtctctg 961 ggaaggtgcg cttcccttcc cacttcagct ctgacttgaa ggacctgctg cggaacctcc 1021 tgcaggtaga tctcaccaag cgctttggga acctcaagaa tggggtcaac gatatcaaga 1081 accacaagtg gtttgccaca actgactgga ttgccatcta ccagaggaag gtggaagctc 1141 ccttcatacc aaagtttaaa ggccctgggg atacgagtaa ctttgacgac tatgaggaag 1201 aagaaatccg ggtctccatc aatgagaagt gtggcaagga gttttctgag ttttaggggc 1261 atgcctgtgc ccccatgggt tttctttttt cttttttctt ttttttggtc gggggggtgg 1321 gagggttgga ttgaacagcc agagggcccc agagttcctt gcatctaatt tcacccccac 1381 cccaccctcc agggttaggg ggagcaggaa gcccagataa tcagagggac agaaacacca 1441 gctgctcccc ctcatcccct tcaccctcct gccccctctc ccacttttcc cttcctcttt 1501 ccccacagcc ccccagcccc tcagccctcc cagcccactt ctgcctgttt taaacgagtt 1561 tctcaactcc agtcagacca ggtcttgctg gtgtatccag ggacagggta tggaaagagg 1621 ggctcacgct taactccagc ccccacccac acccccatcc cacccaacca caggccccac 1681 ttgctaaggg caaatgaacg aagcgccaac cttcctttcg gagtaatcct gcctgggaag 1741 gagagatttt tagtgacatg ttcagtgggt tgcttgctag aattttttta aaaaaacaac 1801 aatttaaaat cttatttaag ttccaccagt gcctccctcc ctccttcctc tactcccacc 1861 cctcccatgt ccccccattc ctcaaatcca ttttaaagag aagcagactg actttggaaa 1921 gggaggcgct ggggtttgaa cctccccgct gctaatctcc cctgggcccc tccccgggga 1981 atcctctctg ccaatcctgc gagggtctag gcccctttag gaagcctccg ctctcttttt 2041 ccccaacaga cctgtcttca cccttgggct ttgaaagcca gacaaagcag ctgcccctct 2101 ccctgccaaa gaggagtcat cccccaaaaa gacagagggg gagccccaag cccaagtctt 2161 tcctcccagc agcgtttccc cccaactcct taattttatt ctccgctaga ttttaacgtc 2221 cagccttccc tcagctgagt ggggagggca tccctgcaaa agggaacaga agaggccaag 2281 tccccccaag ccacggcccg gggttcaagg ctagagctgc tggggagggg ctgcctgttt 2341 tactcaccca ccagcttccg cctcccccat cctgggcgcc cctcctccag cttagctgtc 2401 agctgtccat cacctctccc ccactttctc atttgtgctt ttttctctcg taatagaaaa 2461 gtggggagcc gctggggagc caccccattc atccccgtat ttccccctct cataacttct 2521 ccccatccca ggaggagttc tcaggcctgg ggtggggccc cgggtgggtg cgggggcgat 2581 tcaacctgtg tgctgcgaag gacgagactt cctcttgaac agtgtgctgt tgtaaacata 2641 tttgaaaact attaccaata aagttttgtt taaaaaaaaa aaaaaaaaa

[0036] The Genbank ID for the DNAJB1 gene is 3337. The protein encoded by DNAJB1, has Genebank Accession No. NP 006136 (corresponding nucleotide sequence NM 006145).

[0037] SEQ ID NO: 5 is the DNAJB1 Amino Acid Sequence (NP 006136) (340 aa).

1 MGKDYYQTLG LARGASDEEI KRAYRRQALR YHPDKNKEPG AEEKFKEIAE AYDVLSDPRK 61 REIFDRYGEE GLKGSGPSGG SGGGANGTSF SYTFHGDPHA MFAEFFGGRN PFDTFFGQRN 121 GEEGMDIDDP FSGFPMGMGG FTNVNFGRSR SAQEPARKKQ DPPVTHDLRV SLEEIYSGCT 181 KKMKISHKRL NPDGKSIRNE DKILTIEVKK GWKEGTKITF PKEGDQTSNN IPADIVFVLK 241 DKPHNIFKRD GSDVIYPARI SLREALCGCT VNVPTLDGRT IPWFKDVIR PGMRRKVPGE 301 GLPLPKTPEK RGDLI IEFEV IFPERIPQTS RTVLEQVLPI

[0038] SEQ ID NO: 6 is the DNAJB1 Nucleotide Sequence (NM 006145) (2233 bp):

1 gggacggcga cagcgggtcg gcgggccgca ggagggggtc atgggtaaag actactacca

61 gacgttgggc ctggcccgcg gcgcgtcgga cgaggagatc aagcgggcct accgccgcca

121 ggcgctgcgc taccacccgg acaagaacaa ggagcccggc gccgaggaga agttcaagga

181 gatcgctgag gcctacgacg tgctcagcga cccgcgcaag cgcgagatct tcgaccgcta

241 cggggaggaa ggcctaaagg ggagtggccc cagtggcggt agcggcggtg gtgccaatgg

301 tacctctttc agctacacat tccatggaga ccctcatgcc atgtttgctg agttcttcgg

361 tggcagaaat ccctttgaca ccttttttgg gcagcggaac ggggaggaag gcatggacat

421 tgatgaccca ttctctggct tccctatggg catgggtggc ttcaccaacg tgaactttgg

481 ccgctcccgc tctgcccaag agcccgcccg aaagaagcaa gatcccccag tcacccacga

541 ccttcgagtc tcccttgaag agatctacag cggctgtacc aagaagatga aaatctccca

601 caagcggcta aaccccgacg gaaagagcat tcgaaacgaa gacaaaatat tgaccatcga

661 agtgaagaag gggtggaaag aaggaaccaa aatcactttc cccaaggaag gagaccagac

721 ctccaacaac attccagctg atatcgtctt tgttttaaag gacaagcccc acaatatctt

781 taagagagat ggctctgatg tcatttatcc tgccaggatc agcctccggg aggctctgtg

841 tggctgcaca gtgaacgtcc ccactctgga cggcaggacg atacccgtcg tattcaaaga

901 tgttatcagg cctggcatgc ggcgaaaagt tcctggagaa ggcctccccc tccccaaaac

961 acccgagaaa cgtggggacc tcattattga gtttgaagtg atcttccccg aaaggattcc

1021 ccagacatca agaaccgtac ttgagcaggt tcttccaata tagctatctg agctccccaa

1081 ggactgacca gggacctttc cagagctcaa ggatttctgg acctttctac cagttgtgga

1141 ccatgagagg gtgggagggc ccagggaggg ctttcgtact gctgaatgtt ttccagagca

1201 tatattacaa tctttcaaag tcgcacacta gacttcagtg gtttttcgag ctatagggca

1261 tcaggtggtg ggaacagcag gaaaaggcat tccagtctgc cccactgggt ctggcagccc

1321 tcccgggatg ggcccacatc cacctccagt ccctggccag gggtgagagg cagaccagca

1381 gatggacttg atccctctgt gtctttgggc ttctggctgg tagataatgt caacctgcag

1441 tcttgattcc cagaccctgt acactcctcc ttttctgttg tgtgatcagt ttgtgcttta

1501 ttctgtattt gtctcccatg tcttgctctt ctcctggaga attctgtctt ctctttggcc

1561 atctcaaatt gagaacctaa actattcctg cagaactgcc tggttggcgt ccacaagcaa

1621 tacctctcgt tccagcagga ccaagggagc cagcctccag tgagtgactc cagcaagtgc

1681 agccacctct cccttgatgg tctgggagcc tggcctcagc aaggggcctt cctgacctct

1741 ggctccagtg aagctgaatg tcctcacttt gtgggtcaca ctctttacat ttctgtaagg 1801 caatcttggc acacgtgggg cttaccagtg geccaggtaa ttttttgttt catggactat

1861 ggactctttc aaagggatct gatccttttg aattttgcac agccctagat acaatccctt

1921 ttgataaaag ggtctttgct tctgattaca ggagcactgt ggaacgtctg taaatatgtt

1981 tttataattc catgtatagt tggtgtacac tcaaaacctg tccccggcag ccagtgctct

2041 ctgtataggg ccataatgga attctgaaga aatcttgggg agggaagggg agttggaaca

2101 aatgtctgtt ccctggaggc cagtccagtg ctcagacctt tagactcatt gtaagttgcc

2161 actgccaaca tgagaccaaa gtgtgtgact agtcaatgaa gtgegacage attaaagact

2221 gatgctaaac etc

[0039] SEQ ID NO: 1 is the amino acid sequence of the DNAJB 1 -PRKAC A fusion protein, comprising Exon 1 of DNAJB 1 and Exons 2-10 of PRKACA:

MGKDYYQTLG LARGASDEEI KRAYRRQALR YHPDKNKEPG AEEKFKEIAE AYDVLSDPRK REIFDRYGEE VKEFLAKAKE DFLKKWESPA QNTAHLDQFE RIKTLGTGSF GRVMLVKHKE TGNHYAMKIL DKQKVVKLKQ IEHTLNEKRI LQAVNFPFLV KLEFSFKDNS NLYMVMEYVP GGEMFSHLRR IGRFSEPHAR FYAAQIVLTF EYLHSLDLIY RDLKPENLLI DQQGYIQVTD FGFAKRVKGR TWTLCGTPEY LAPEIILSKG YNKAVDWWAL GVLIYEMAAG YPPFFADQPI QIYEKIVSGK VRFPSHFSSD LKDLLRNLLQ VDLTKRFGNL KNGVNDIKNH KWFATTDWIA IYQRKVEAPF I PKFKGPGDT SNFDDYEEEE IRVSINEKCG KEFSEF

[0040] SEQ ID NO : 2 is the nucleotide sequence encoding the DNAJB 1 -PRKACA fusion protein, comprising Exon 1 of DNAJB 1 and Exons 2-10 of PRKACA:

ATGGGTAAAGACTACTACCAGACGTTGGGCCTGGCCCGCGGCGCGTCGGACGAGGAGAT CAAGCGGGCCTACCGCCGCCAGGCGCTGCGCTACCACCCGGACAAGAACAAGGAGCCCG GCGCCGAGGAGAAGTTCAAGGAGATCGCTGAGGCCTACGACGTGCTCAGCGACCCGCGC AAGCGCGAGATCTTCGACCGCTACGGGGAGGAAGTGAAAGAATTCTTAGCCAAAGCCAA AGAAGATTTTCTTAAAAAATGGGAAAGTCCCGCTCAGAACACAGCCCACTTGGATCAGT TTGAACGAATCAAGACCCTCGGCACGGGCTCCTTCGGGCGGGTGATGCTGGTGAAACAC AAGGAGACCGGGAACCACTATGCCATGAAGATCCTCGACAAACAGAAGGTGGTGAAACT GAAACAGATCGAACACACCCTGAATGAAAAGCGCATCCTGCAAGCTGTCAACTTTCCGT TCCTCGTCAAACTCGAGTTCTCCTTCAAGGACAACTCAAACTTATACATGGTCATGGAG TACGTGCCCGGCGGGGAGATGTTCTCACACCTACGGCGGATCGGAAGGTTCAGTGAGCC CCATGCCCGTTTCTACGCGGCCCAGATCGTCCTGACCTTTGAGTATCTGCACTCGCTGG ATCTCATCTACAGGGACCTGAAGCCGGAGAATCTGCTCATTGACCAGCAGGGCTACATT CAGGTGACAGACTTCGGTTTCGCCAAGCGCGTGAAGGGCCGCACTTGGACCTTGTGCGG CACCCCTGAGTACCTGGCCCCTGAGATTATCCTGAGCAAAGGCTACAACAAGGCCGTGG ACTGGTGGGCCCTGGGGGTTCTTATCTATGAAATGGCCGCTGGCTACCCGCCCTTCTTC GCAGACCAGCCCATCCAGATCTATGAGAAGATCGTCTCTGGGAAGGTGCGCTTCCCTTC CCACTTCAGCTCTGACTTGAAGGACCTGCTGCGGAACCTCCTGCAGGTAGATCTCACCA AGCGCTTTGGGAACCTCAAGAATGGGGTCAACGATATCAAGAACCACAAGTGGTTTGCC ACAACTGACTGGATTGCCATCTACCAGAGGAAGGTGGAAGCTCCCTTCATACCAAAGTT TAAAGGCCCTGGGGATACGAGTAACTTTGACGACTATGAGGAAGAAGAAATCCGGGTCT CCATCAATGAGAAGTGTGGCAAGGAGTTTTCTGAGTTTTAG

[0041] As used herein, a "DNA J-cAMP-dependent PK fusion molecule" can be a nucleic acid (e.g., synthetic, purified, and/or recombinant) which encodes a polypeptide corresponding to a fusion protein comprising a heat shock protein fused to a kinase domain of a cAMP-dependent protein kinase. It can also be a fusion protein comprising a J domain fused to a kinase domain of a cAMP-dependent protein kinase. The molecule can further be a fusion protein comprising a DNAJB1 protein fused to a kinase domain of a cAMP-dependent protein kinase. For example, a DNA J-cAMP-dependent PK fusion molecule can include a DNAJB1-PRKACA fusion protein (e.g., comprising the amino acid sequence shown in SEQ ID NO: 1, or comprising the nucleic acid sequence shown in SEQ ID NO: 2). The DNA J-cAMP-dependent PK fusion molecule can also comprise the amino acid sequence shown in SEQ ID NOS: 3 and 5, or comprising the nucleic acid sequence shown in SEQ ID NOS: 4 and 6). For example, a DNA J-cAMP-dependent PK fusion molecule can comprise the amino acid sequence corresponding to Genebank Accession no.

NP 997401; or the nucleotide sequence corresponding to Genebank Accession no. NM 207518. A DNA J-cAMP-dependent PK fusion molecule can include a variant of the above described examples, such as a fragment thereof.

[0042] The nucleic acid can be any type of nucleic acid, including genomic DNA,

complementary DNA (cDNA), recombinant DNA, synthetic or semi-synthetic DNA, as well as any form of corresponding RNA. A cDNA is a form of DNA artificially synthesized from a messenger RNA template and is used to produce gene clones. A synthetic DNA is free of modifications that can be found in cellular nucleic acids, including, but not limited to, histones and methylation. For example, a nucleic acid encoding a DNA J-cAMP-dependent PK fusion molecule can comprise a recombinant nucleic acid encoding such a protein. The nucleic acid can be a non-naturally occurring nucleic acid created artificially (such as by assembling, cutting, ligating or amplifying sequences). It can be double-stranded or single-stranded.

[0043] The invention further provides for nucleic acids that are complementary to a DNA J- cAMP-dependent PK fusion molecule. Complementary nucleic acids can hybridize to the nucleic acid sequence described above under stringent hybridization conditions. Non- limiting examples of stringent hybridization conditions include temperatures above 30°C, above 35°C, in excess of 42°C, and/or salinity of less than about 500 mM, or less than 200 mM. Hybridization conditions can be adjusted by the skilled artisan via modifying the temperature, salinity and/or the

concentration of other reagents such as SDS or SSC.

[0044] According to the invention, protein variants can include amino acid sequence modifications. For example, amino acid sequence modifications fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions can include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues. Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. These variants ordinarily are prepared by site-specific mutagenesis of nucleotides in the DNA encoding the protein, thereby producing DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture.

[0045] In one embodiment, a DNA J-cAMP-dependent PK fusion molecule comprises a protein or polypeptide encoded by a nucleic acid sequence encoding a DNA J-cAMP-dependent PK fusion molecule, such as the sequences shown in SEQ ID NO: 2. In some embodiments, the nucleic acid sequence encoding a DNA J-cAMP-dependent PK fusion molecule is about 70%, about 75%, about 80%, about 85%, about 90%, about 93%, about 95%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 2. In another embodiment, the polypeptide can be modified, such as by glycosylations and/or acetylations and/or chemical reaction or coupling, and can contain one or several non-natural or synthetic amino acids. An example of a DNA J-cAMP-dependent PK fusion molecule is the polypeptide having the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the DNA J-cAMP-dependent PK fusion molecule that is a polypeptide is about 70%>, about 75%, about 80%, about 85%, about 90%, about 93%, about 95%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 1. In another embodiment, a DNA J-cAMP-dependent PK fusion molecule can be a fragment of a DNA J-cAMP-dependent PK fusion protein. For example, the DNA J-cAMP-dependent PK fusion molecule can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NOS: 1. The fragment can comprise at least about 10 amino acids, a least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, a least about 50 amino acids, at least about 60 amino acids, or at least about 75 amino acids of SEQ ID NOS: 1. Fragments include all possible amino acid lengths between about 8 and 100 about amino acids, for example, lengths between about 10 and 100 amino acids, between about 15 and 100 amino acids, between about 20 and 100 amino acids, between about 35 and 100 amino acids, between about 40 and 100 amino acids, between about 50 and 100 amino acids, between about 70 and 100 amino acids, between about 75 and 100 amino acids, or between about 80 and 100 amino acids. Fragments include all possible amino acid lengths between about 100 and 400 amino acids, for example, lengths between about 125 and 400 amino acids, between about 150 and 400 amino acids, between about 175 and 400 amino acids, between about 200 and 400 amino acids, between about 225 and 400 amino acids, between about 250 and 400 amino acids, between about 275 and 400 amino acids, between about 300 and 400 amino acids, between about 325 and 400 amino acids, between about 350 and 400 amino acids, Or between about 375 and 400 amino acids. [0046] Chemical Synthesis. Nucleic acid sequences encoding a DNA J-cAMP-dependent PK fusion molecule can be synthesized, in whole or in part, using chemical methods known in the art. Alternatively, a polypeptide can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques. Protein synthesis can either be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431 A Peptide Synthesizer (Perkin Elmer).

[0047] Optionally, polypeptides fragments can be separately synthesized and combined using chemical methods to produce a full-length molecule. For example, these methods can be utilized to synthesize a fusion protein of the invention. In one embodiment, the fusion protein comprises a heat shock protein fused to a kinase domain of a cAMP-dependent protein kinase. In another embodiment, the fusion protein comprises a J domain fused to a kinase domain of a cAMP- dependent protein kinase. In a further embodiment, the fusion protein comprises a DNAJB1 protein fused to a kinase domain of a cAMP-dependent protein kinase. In some embodiments, the fusion protein comprises the DNAJB1-PRKACA fusion protein. For example, the DNAJB1- PRKACA fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1.

[0048] Obtaining, Purifying and Detecting DNA J-cAMP-dependent PK fusion molecules. A polypeptide encoded by a nucleic acid, such as a nucleic acid encoding a DNA J-cAMP-dependent PK fusion molecule, or a variant thereof, can be obtained by purification from human cells expressing a protein or polypeptide encoded by such a nucleic acid. Non-limiting purification methods include size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis.

[0049] A synthetic polypeptide can be substantially purified via high performance liquid chromatography (HPLC), such as ion exchange chromatography (IEX-HPLC). The composition of a synthetic polypeptide, such as a DNA J-cAMP-dependent PK fusion molecule, can be confirmed by amino acid analysis or sequencing.

[0050] Other constructions can also be used to join a nucleic acid sequence encoding a polypeptide/protein of the claimed invention to a nucleotide sequence encoding a polypeptide domain which will facilitate purification of soluble proteins. Such purification facilitating domains include, but are not limited to, metal chelating peptides such as histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on

immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity

purification system (Immunex Corp., Seattle, Wash.). Including cleavable linker sequences (i.e., those specific for Factor Xa or enterokinase (Invitrogen, San Diego, Calif.)) between the purification domain and a polypeptide encoded by a nucleic acid of the invention also can be used to facilitate purification. For example, the skilled artisan can use an expression vector encoding 6 histidine residues that precede a thioredoxin or an enterokinase cleavage site in conjunction with a nucleic acid of interest. The histidine residues facilitate purification by immobilized metal ion affinity chromatography, while the enterokinase cleavage site provides a means for purifying the polypeptide encoded by, for example, an DNA J-cAMP-dependent PK fusion molecule, such as DNAJB 1 -PRKACA.

[0051] Host cells which contain a nucleic acid encoding a DNA J-cAMP-dependent PK fusion molecule, and which subsequently express the same, can be identified by various procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein. For example, the presence of a nucleic acid encoding a DNA J-cAMP-dependent PK fusion molecule can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments of nucleic acids encoding the same. In one embodiment, a nucleic acid fragment of a DNA J-cAMP-dependent PK fusion molecule can encompass any portion of at least about 8 consecutive nucleotides of SEQ ID NO: 2. In another embodiment, the fragment can comprise at least about 10 consecutive nucleotides, at least about 15 consecutive nucleotides, at least about 20 conseutive nucleotides, or at least about 30 consecutive nucleotides of SEQ ID NOS: 2. Fragments can include all possible nucleotide lengths between about 8 and about 100 nucleotides, for example, lengths between about 15 and about 100 nucleotides, or between about 20 and about 100 nucleotides. Nucleic acid amplification-based assays involve the use of

oligonucleotides selected from sequences encoding a DNA J-cAMP-dependent PK fusion molecule nucleic acid, or DNA J-cAMP-dependent PK fusion molecule nucleic acid to detect transformants which contain a nucleic acid encoding a protein or polypeptide of the same.

[0052] Protocols are known in the art for detecting and measuring the expression of a polypeptide encoded by a nucleic acid, such as a nucleic acid encoding a DNA J-cAMP-dependent PK fusion molecule, using either polyclonal or monoclonal antibodies specific for the polypeptide. Non-limiting examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based

immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on a polypeptide encoded by a nucleic acid, such as a nucleic acid encoding a DNA J-cAMP-dependent PK fusion molecule, can be used, or a competitive binding assay can be employed.

[0053] Labeling and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and amino acid assays. Methods for producing labeled hybridization or PCR probes for detecting sequences related to nucleic acid sequences encoding a protein, such as DNA J-cAMP-dependent PK fusion molecule, include, but are not limited to, oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide. Alternatively, nucleic acid sequences, such as nucleic acids encoding a DNA J-cAMP-dependent PK fusion molecule, can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical). Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, and/or magnetic particles.

[0054] A fragment can be a fragment of a protein, such as a DNA J-cAMP-dependent PK fusion protein. For example, a fragment of a DNA J-cAMP-dependent PK fusion molecule can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NO: 1. The fragment can comprise at least about 10 consecutive amino acids, at least about 20 consecutive amino acids, at least about 30 consecutive amino acids, at least about 40 consecutive amino acids, a least about 50 consecutive amino acids, at least about 60 consecutive amino acids, at least about 70 consecutive amino acids, at least about 75 consecutive amino acids, at least about 80 consecutive amino acids, at least about 85 consecutive amino acids, at least about 90 consecutive amino acids, at least about 95 consecutive amino acids, at least about 100 consecutive amino acids, at least about 150 consecutive amino acids, at least about 200 consecutive amino acids, at least about 250 consecutive amino acids, at least about 300 consecutive amino acids, at least about 350, or at least about 400 consecutive amino acids of SEQ ID NO: 1. Fragments include all possible amino acid lengths between about 8 and 100 about amino acids, for example, lengths between about 10 and about 100 amino acids, between about 15 and about 100 amino acids, between about 20 and about 100 amino acids, between about 35 and about 100 amino acids, between about 40 and about 100 amino acids, between about 50 and about 100 amino acids, between about 70 and about 100 amino acids, between about 75 and about 100 amino acids, or between about 80 and about 100 amino acids. Cell Transfection

[0055] Host cells transformed with a nucleic acid sequence of interest can be cultured under conditions suitable for the expression and recovery of the protein from cell culture. The

polypeptide produced by a transformed cell can be secreted or contained intracellularly depending on the sequence and/or the vector used. Expression vectors containing a nucleic acid sequence, such as a nucleic acid encoding a DNA J-cAMP-dependent PK fusion molecule, can be designed to contain signal sequences which direct secretion of soluble polypeptide molecules encoded by the nucleic acid. Cell transfection and culturing methods are described in more detail below.

[0056] A eukaryotic expression vector can be used to transfect cells in order to produce proteins encoded by nucleotide sequences of the vector, e.g. those encoding a DNA J-cAMP- dependent PK fusion molecule. Mammalian cells can contain an expression vector (for example, one that contains a nucleic acid encoding a fusion protein comprising a heat shock protein fused to a kinase domain of a cAMP-dependent protein kinase; a nucleic acid encoding a fusion protein comprising a J domain fused to a kinase domain of a cAMP-dependent protein kinase; a nucleic acid encoding a fusion protein comprising a DNAJB 1 protein fused to a kinase domain of a cAMP- dependent protein kinase; or a nucleic acid encoding a fusion protein comprising DNAJB 1- PRKACA fusion protein via introducing the expression vector into an appropriate host cell via methods known in the art.

[0057] A host cell strain can be chosen for its ability to modulate the expression of the inserted sequences or to process the expressed polypeptide encoded by a nucleic acid, in the desired fashion. Such modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing which cleaves a "prepro" form of the polypeptide also can be used to facilitate correct insertion, folding and/or function. Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available from the American Type Culture Collection (ATCC; 10801 University Boulevard, Manassas, Va. 20110-2209) and can be chosen to ensure the correct modification and processing of the foreign protein.

[0058] An exogenous nucleic acid can be introduced into a cell via a variety of techniques known in the art, such as lipofection, microinjection, calcium phosphate or calcium chloride precipitation, DEAE-dextran-mediated transfection, or electroporation. Electroporation is carried out at approximate voltage and capacitance to result in entry of the DNA construct(s) into cells of interest (such as glioma cells (cell line SF188), neuroblastoma cells (cell lines IMR-32, SK-N-SH, SH-F and SH-N), astrocytes and the like). Other transfection methods also include modified calcium phosphate precipitation, polybrene precipitation, liposome fusion, and receptor-mediated gene delivery.

[0059] Cells that will be genetically engineered can be primary and secondary cells obtained from various tissues, and include cell types which can be maintained and propagated in culture. Non-limiting examples of primary and secondary cells include epithelial cells, neural cells, endothelial cells, glial cells, fibroblasts, muscle cells (such as myoblasts) keratinocytes, formed elements of the blood (e.g., lymphocytes, bone marrow cells), and precursors of these somatic cell types.

[0060] Vertebrate tissue can be obtained by methods known to one skilled in the art, such a punch biopsy or other surgical methods of obtaining a tissue source of the primary cell type of interest. In one embodiment, a punch biopsy or removal (e.g., by aspiration) can be used to obtain a source of cancer cells (for example, hepatic cells, hepatoblastoma cells, and hepatocellular carcinoma cells). A mixture of primary cells can be obtained from the tissue, using methods readily practiced in the art, such as explanting or enzymatic digestion (for examples using enzymes such as pronase, trypsin, collagenase, elastase dispase, and chymotrypsin). Biopsy methods have also been described in United States Patent No. 7,419,661 and PCT application publication WO 2001/32840, and each are hereby incorporated by reference.

[0061] Primary cells can be acquired from the individual to whom the genetically engineered primary or secondary cells are administered. However, primary cells can also be obtained from a donor, other than the recipient, of the same species. The cells can also be obtained from another species (for example, rabbit, cat, mouse, rat, sheep, goat, dog, horse, cow, bird, or pig). Primary cells can also include cells from a purified vertebrate tissue source grown attached to a tissue culture substrate (for example, flask or dish) or grown in a suspension; cells present in an explant derived from tissue; both of the aforementioned cell types plated for the first time; and cell culture suspensions derived from these plated cells. Secondary cells can be plated primary cells that are removed from the culture substrate and replated, or passaged, in addition to cells from the subsequent passages. Secondary cells can be passaged one or more times. These primary or secondary cells can contain expression vectors having a gene that encodes a DNA J-cAMP- dependent PK fusion molecule.

Cell Culturing [0062] Various culturing parameters can be used with respect to the host cell being cultured. Appropriate culture conditions for mammalian cells are well known in the art (Cleveland WL, et al, J Immunol Methods, 1983, 56(2): 221-234) or can be determined by the skilled artisan (see, for example, Animal Cell Culture: A Practical Approach 2nd Ed., Rickwood, D. and Hames, B. D., eds. (Oxford University Press: New York, 1992)). Cell culturing conditions can vary according to the type of host cell selected. Commercially available medium can be utilized. Non-limiting examples of medium include, for example, Minimal Essential Medium (MEM, Sigma, St. Louis, Mo.); Dulbecco's Modified Eagles Medium (DMEM, Sigma); Ham's F10 Medium (Sigma);

HyClone cell culture medium (HyClone, Logan, Utah); RPMI-1640 Medium (Sigma); and chemically-defined (CD) media, which are formulated for various cell types, e.g., CD-CHO

Medium (Invitrogen, Carlsbad, Calif).

[0063] The cell culture media can be supplemented as necessary with supplementary components or ingredients, including optional components, in appropriate concentrations or amounts, as necessary or desired. Cell culture medium solutions provide at least one component from one or more of the following categories: (1) an energy source, usually in the form of a carbohydrate such as glucose; (2) all essential amino acids, and usually the basic set of twenty amino acids plus cysteine; (3) vitamins and/or other organic compounds required at low

concentrations; (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that can be required at very low concentrations, usually in the micromolar range.

[0064] The medium also can be supplemented electively with one or more components from any of the following categories: (1) salts, for example, magnesium, calcium, and phosphate; (2) hormones and other growth factors such as, serum, insulin, transferrin, and epidermal growth factor; (3) protein and tissue hydrolysates, for example peptone or peptone mixtures which can be obtained from purified gelatin, plant material, or animal byproducts; (4) nucleosides and bases such as, adenosine, thymidine, and hypoxanthine; (5) buffers, such as HEPES; (6) antibiotics, such as gentamycin or ampicillin; (7) cell protective agents, for example pluronic polyol; and (8) galactose. In one embodiment, soluble factors can be added to the culturing medium.

[0065] The mammalian cell culture that can be used with the present invention is prepared in a medium suitable for the type of cell being cultured. In one embodiment, the cell culture medium can be any one of those previously discussed (for example, MEM) that is supplemented with serum from a mammalian source (for example, fetal bovine serum (FBS)). In another embodiment, the medium can be a conditioned medium to sustain the growth of host cells. [0066] Three-dimensional cultures can be formed from agar (such as Gey's Agar), hydrogels (such as matrigel, agarose, and the like; Lee et al, (2004) Biomaterials 25: 2461-2466) or polymers that are cross-linked. These polymers can comprise natural polymers and their derivatives, synthetic polymers and their derivatives, or a combination thereof. Natural polymers can be anionic polymers, cationic polymers, amphipathic polymers, or neutral polymers. Non-limiting examples of anionic polymers can include hyaluronic acid, alginic acid (alginate), carageenan, chondroitin sulfate, dextran sulfate, and pectin. Some examples of cationic polymers, include but are not limited to, chitosan or polylysine. (Peppas et al, (2006) Adv Mater. 18: 1345-60; Hoffman, A. S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NY Acad Sci 944: 62-73). Examples of amphipathic polymers can include, but are not limited to collagen, gelatin, fibrin, and carboxymethyl chitin. Non-limiting examples of neutral polymers can include dextran, agarose, or pullulan. (Peppas et al, (2006) Adv Mater. 18: 1345-60; Hoffman, A. S., (2002) Adv Drug Deliv Rev. 43: 3-12; Hoffman, A. S., (2001) Ann NY Acad Sci 944: 62-73).

[0067] Cells to be cultured can harbor introduced expression vectors, such as plasmids. The expression vector constructs can be introduced via transformation, microinjection, transfection, lipofection, electroporation, or infection. The expression vectors can contain coding sequences, or portions thereof, encoding the proteins for expression and production. Expression vectors containing sequences encoding the produced proteins and polypeptides, as well as the appropriate transcriptional and translational control elements, can be generated using methods well known to and practiced by those skilled in the art. These methods include synthetic techniques, in vitro recombinant DNA techniques, and in vivo genetic recombination which are described in J.

Sambrook et al., 2001, Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y. and in F. M. Ausubel et al., 1989, Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y.

DNA J-cAMP-dependent PK fusion Molecule Inhibitors

[0068] The invention provides methods for use of compounds that decrease the expression level or activity of a DNA J-cAMP-dependent PK fusion molecule in a subject. In addition, the invention provides methods for using compounds for the treatment of a gene-fusion associated cancer. In one embodiment, the gene-fusion associated cancer is an epithelial cancer. In one embodiment, the gene-fusion associated cancer comprises glioblastoma multiforme, breast cancer, lung cancer, prostate cancer, or colorectal carcinoma. [0069] As used herein, a "DNA J-cAMP-dependent PK fusion molecule inhibitor" refers to a compound that interacts with a DNA J-cAMP-dependent PK fusion molecule of the invention and modulates its activity and/or its expression. For example, the compound can decrease the activity or expression of a DNA J-cAMP-dependent PK fusion molecule. The compound can be an antagonist of a DNA J-cAMP-dependent PK fusion molecule (e.g., a DNA J-cAMP-dependent PK fusion molecule inhibitor). Some non- limiting examples of DNA J-cAMP-dependent PK fusion molecule inhibitors include peptides (such as peptide fragments comprising a DNA J-cAMP- dependent PK fusion molecule, or antibodies or fragments thereof), small molecules, and nucleic acids (such as siRNA or antisense RNA specific for a nucleic acid comprising a DNA J-cAMP- dependent PK fusion molecule). Antagonists of a DNA J-cAMP-dependent PK fusion molecule decrease the amount or the duration of the activity of a DNA J-cAMP-dependent PK fusion protein. In one embodiment, the fusion protein comprises a heat shock protein fused to a kinase domain of a cAMP-dependent protein kinase. In another embodiment, the fusion protein comprises a J domain fused to a kinase domain of a cAMP-dependent protein kinase. In a further embodiment, the fusion protein comprises a DNAJB1 protein fused to a kinase domain of a cAMP-dependent protein kinase. In some embodiments, the fusion protein comprises DNAJBl-PRKACA fusion protein. Antagonists include proteins, nucleic acids, antibodies, small molecules, or any other molecule which decrease the activity of a DNA J-cAMP-dependent PK fusion molecule.

[0070] The term "modulate," as it appears herein, refers to a change in the activity or expression of a DNA J-cAMP-dependent PK fusion molecule. For example, modulation can cause a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of a DNA J-cAMP-dependent PK fusion molecule, such as a DNA J- cAMP-dependent PK fusion protein.

[0071] In one embodiment, a DNA J-cAMP-dependent PK fusion molecule inhibitor can be a peptide fragment of a DNA J-cAMP-dependent PK fusion protein that binds to the protein itself.

[0072] For example, the DNA J-cAMP-dependent PK fusion polypeptide can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NO: 1. The fragment can comprise at least about 10 consecutive amino acids, at least about 20 consecutive amino acids, at least about 30 consecutive amino acids, at least about 40 consecutive amino acids, a least about 50 consecutive amino acids, at least about 60 consecutive amino acids, at least about 70 consecutive amino acids, at least about 75 consecutive amino acids, at least about 80 consecutive amino acids, at least about 85 consecutive amino acids, at least about 90 consecutive amino acids, at least about 95

consecutive amino acids, at least about 100 consecutive amino acids, at least about 150 consecutive amino acids, at least about 200 consecutive amino acids, at least about 250 consecutive amino acids, at least about 300 consecutive amino acids, at least about 350 consecutive amino acids, or at least about 400 consecutive amino acids of SEQ ID NO: 1. Fragments include all possible amino acid lengths between about 8 and 100 about amino acids, for example, lengths between about 10 and about 100 amino acids, between about 15 and about 100 amino acids, between about 20 and about 100 amino acids, between about 35 and about 100 amino acids, between about 40 and about 100 amino acids, between about 50 and about 100 amino acids, between about 70 and about 100 amino acids, between about 75 and about 100 amino acids, or between about 80 and about 100 amino acids. These peptide fragments can be obtained commercially or synthesized via liquid phase or solid phase synthesis methods (Atherton et al, (1989) Solid Phase Peptide Synthesis: a Practical Approach. IRL Press, Oxford, England). The DNA J-cAMP-dependent PK fusion peptide fragments can be isolated from a natural source, genetically engineered, or chemically prepared. These methods are well known in the art.

[0073] A DNA J-cAMP-dependent PK fusion molecule inhibitor can be a protein, such as an antibody (monoclonal, polyclonal, humanized, chimeric, or fully human), or a binding fragment thereof, directed against a DNA J-cAMP-dependent PK fusion moleculeof the invention. An antibody fragment can be a form of an antibody other than the full-length form and includes portions or components that exist within full-length antibodies, in addition to antibody fragments that have been engineered. Antibody fragments can include, but are not limited to, single chain Fv (scFv), diabodies, Fv, and (Fab')₂, triabodies, Fc, Fab, CDR1, CDR2, CDR3, combinations of CDR's, variable regions, tetrabodies, bifunctional hybrid antibodies, framework regions, constant regions, and the like (see, Maynard et al, (2000) Ann. Rev. Biomed. Eng. 2:339-76; Hudson (1998) Curr. Opin. Biotechnol. 9:395-402). Antibodies can be obtained commercially, custom generated, or synthesized against an antigen of interest according to methods established in the art (see United States Patent Nos. 6,914,128, 5,780,597, 5,811,523; Roland E. Kontermann and Stefan Dubel (editors), Antibody Engineering, Vol. I & II. (2010) 2^nd ed., Springer; Antony S. Dimitrov (editor), Therapeutic Antibodies: Methods and Protocols (Methods in Molecular Biology), (2009), Humana Press; Benny Lo (editor) Antibody Engineering: Methods and Protocols (Methods in Molecular Biology), (2004) Humana Press, each of which are hereby incorporated by reference in their entireties). For example, antibodies directed to a DNA J-cAMP-dependent PK fusion molecule can be obtained commercially from Abeam, Santa Cruz Biotechnology, Abgent, R&D Systems, Novus Biologicals, etc. Human antibodies directed to a DNA J-cAMP-dependent PK fusion molecule (such as monoclonal, humanized, fully human, or chimeric antibodies) can be useful antibody therapeutics for use in humans. In one embodiment, an antibody or binding fragment thereof is directed against SEQ ID NOS: 1 , 3, or 5.

[0074] Inhibition of R A encoding a DNA J-cAMP-dependent PK fusion molecule can effectively modulate the expression of a DNA J-cAMP-dependent PK fusion molecule. Inhibitors are selected from the group comprising: siRNA; interfering RNA or RNAi; dsRNA; RNA

Polymerase III transcribed DNAs; ribozymes; and antisense nucleic acids, which can be RNA, DNA, or an artificial nucleic acid.

[0075] Antisense oligonucleotides, including antisense DNA, RNA, and DNA/RNA molecules, act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the DNA sequence encoding a DNA J-cAMP-dependent PK fusion molecule can be synthesized, e.g., by conventional phosphodiester techniques (Dallas et al, (2006) Med. Sci. om^'i.l2(4):RA67-74; Kalota et al, (2006) Handb. Exp. Pharmacol. 173: 173-96; Lutzelburger et al, (2006) Handb. Exp. Pharmacol. 173:243-59). Antisense nucleotide sequences include, but are not limited to: morpho linos, 2'-0-methyl polynucleotides, DNA, RNA and the like.

[0076] siRNA comprises a double stranded structure containing from about 15 to about 50 base pairs, for example from about 21 to about 25 base pairs, and having a nucleotide sequence identical or nearly identical to an expressed target gene or RNA within the cell. The siRNA comprise a sense RNA strand and a complementary antisense RNA strand annealed together by standard Watson-Crick base-pairing interactions. The sense strand comprises a nucleic acid sequence which is substantially identical to a nucleic acid sequence contained within the target miRNA molecule. "Substantially identical" to a target sequence contained within the target mRNA refers to a nucleic acid sequence that differs from the target sequence by about 3% or less. The sense and antisense strands of the siRNA can comprise two complementary, single-stranded RNA molecules, or can comprise a single molecule in which two complementary portions are base-paired and are covalently linked by a single-stranded "hairpin" area. See also, McMnaus and Sharp (2002) Nat Rev Genetics, 3:737-47, and Sen and Blau (2006) FASEB J., 20: 1293-99, the entire disclosures of which are herein incorporated by reference.

[0077] The siRNA can be altered RNA that differs from naturally-occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations can include addition of non-nucleotide material, such as to the end(s) of the siRNA or to one or more internal nucleotides of the siRNA, or modifications that make the siRNA resistant to nuclease digestion, or the substitution of one or more nucleotides in the siRNA with deoxyribo-nucleotides. One or both strands of the siRNA can also comprise a 3' overhang. As used herein, a 3' overhang refers to at least one unpaired nucleotide extending from the 3 '-end of a duplexed RNA strand. For example, the siRNA can comprise at least one 3' overhang of from 1 to about 6 nucleotides (which includes ribonucleotides or deoxyribonucleotides) in length, or from 1 to about 5 nucleotides in length, or from 1 to about 4 nucleotides in length, or from about 2 to about 4 nucleotides in length. For example, each strand of the siRNA can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu").

[0078] siRNA can be produced chemically or biologically, or can be expressed from a recombinant plasmid or viral vector (for example, see U.S. Patent No. 7,294,504 and U.S. Patent No. 7,422,896, the entire disclosures of which are herein incorporated by reference). Exemplary methods for producing and testing dsRNA or siRNA molecules are described in U.S. Patent Application Publication No. 2002/0173478 to Gewirtz, U.S. Patent No. 8,071,559 to Hannon et al, and in U.S. Patent No. 7,148,342 to Tolentino et al., the entire disclosures of which are herein incorporated by reference.

[0079] In one embodiment, an siRNA directed to a human nucleic acid sequence comprising a DNA J-cAMP-dependent PK fusion molecule can be generated SEQ ID NO: 2. In another embodiment, an siRNA directed to a human nucleic acid sequence comprising a breakpoint of anDNA J-cAMP-dependent PK fusion molecule can be generated against SEQ ID NO: 2.

[0080] RNA polymerase III transcribed DNAs contain promoters, such as the U6 promoter. These DNAs can be transcribed to produce small hairpin RNAs in the cell that can function as siRNA or linear RNAs, which can function as antisense RNA. The DNA J-cAMP-dependent PK fusion molecule inhibitor can comprise ribonucleotides, deoxyribonucleotides, synthetic

nucleotides, or any suitable combination such that the target RNA and/or gene is inhibited. In addition, these forms of nucleic acid can be single, double, triple, or quadruple stranded, (see for example Bass (2001) Nature, 411 :428-429; Elbashir et al, (2001) Nature, 411 :494 498; U.S. Patent No. 6,509,154; U.S. Patent Application Publication No. 2003/0027783; and PCT Publication Nos. WO 00/044895, WO 99/032619, WO 00/01846, WO 01/029058, WO 00/044914).

[0081] A DNA J-cAMP-dependent PK fusion molecule inhibitor can be a small molecule that binds to a DNA J-cAMP-dependent PK fusion protein described herein and disrupts its function. Small molecules are a diverse group of synthetic and natural substances generally having low molecular weights. They can be isolated from natural sources (for example, plants, fungi, microbes and the like), are obtained commercially and/or available as libraries or collections, or synthesized. Candidate small molecules that inhibit a DNA J-cAMP-dependent PK fusion protein can be identified via in silico screening or high-through-put (HTP) screening of combinatorial libraries according to methods established in the art (e.g., see Potyrailo et al, (2011) ACS Comb Sci.

13(6):579-633; Mensch et al, (2009) JPharm Sci. 98(12):4429-68; Schnur (2008) Curr Opin Drug Discov Devel. 11(3):375-80; and Jhoti (2007) Ernst Schering Found Symp Proc. (3): 169-85, each of which are hereby incorporated by reference in their entireties.) Most conventional

pharmaceuticals, such as aspirin, penicillin, and many chemotherapeutics, are small molecules, can be obtained commercially, can be chemically synthesized, or can be obtained from random or combinatorial libraries as described below {see, e.g., Werner et al, (2006) Brief Fund. Genomic Proteomic 5(l):32-6).

[0082] Non- limiting examples of DNA J-cAMP-dependent PK fusion molecule inhibitors include the PKA inhibitors Rp-8-PIP-cAMPS (Santa Cruz Biotechnology cat. # sc-391036);

Adenosine 3',5'-cyclic Monophosphorothioate, 2'-0-Monobutyryl-, Rp-Isomer, Sodium Salt (Santa Cruz Biotechnology cat. # sc-391041); 4-Cyano-3-methylisoquinoline (Santa Cruz Biotechnology cat. # sc-391037); 5-Iodotubercidin (Santa Cruz Biotechnology cat. # sc-3531); 8-Bromo-2'- monobutyryladenosine-3 ',5 '-cyclic monophosphorothioate, Rp-isomer (Santa Cruz Biotechnology cat. # sc-391035); A-3 Hydrochloride (Santa Cruz Biotechnology cat. # sc-221177); KT 5720 (Santa Cruz Biotechnology cat. # sc-3538); ML-9 (Santa Cruz Biotechnology cat. # sc-200519); Adenosine 3',5'-cyclic Monophosphorothioate, 8-Chloro-, Rp-Isomer, Sodium Salt (Santa Cruz Biotechnology cat. # sc- 391027); H-89, Dihydrochloride (Santa Cruz Biotechnology cat. # sc- 3537); Rp-8-Hexylaminoadenosine 3 ',5 '-monophosphorothioate (Santa Cruz Biotechnology cat. # sc- 391034);|Bisindolylmaleimide IV (Santa Cruz Biotechnology cat. # sc-3543); Rp-Adenosine 3 ',5 '-cyclic Monophosphorothioate, Sodium Salt and E-7080 (Santa Cruz Biotechnology cat. # sc- 202038); H-8 · 2HCL (Santa Cruz Biotechnology cat. # sc-202038); HA- 1004 (Santa Cruz Biotechnology cat. # sc-200537); CGP 74514A (Santa Cruz Biotechnology cat. # sc-391004); Calphostin C (Santa Cruz Biotechnology cat. # sc-3545); PKI (5-24), PKA Inhibitor (Santa Cruz Biotechnology cat. # sc-201159); H-9 hydrochloride (Santa Cruz Biotechnology cat. # sc-200553); daphnetin (Santa Cruz Biotechnology cat. # sc-203022); HA- 1004 hydrochloride|(Santa Cruz Biotechnology cat. # sc-391033); HA- 1077 dihydrochloride (Santa Cruz Biotechnology cat. # sc- 200583); K-252a (Santa Cruz Biotechnology cat. # sc-200517); K-252b (Santa Cruz Biotechnology cat. # sc-200585); Rp-cAMPS (Santa Cruz Biotechnology cat. # sc-24010); HA- 100

dihydrochloride (Santa Cruz Biotechnology cat. # sc-203072); Myricetin (Santa Cruz

Biotechnology cat. # sc-203147); K252c (Santa Cruz Biotechnology cat. # sc-24011); ML-7 (Santa Cruz Biotechnology cat. # sc-200557); PKA inhibitor (Santa Cruz Biotechnology cat. # sc-3010); H-7 (Santa Cruz Biotechnology cat. # sc-215123); Rp-8-CPT-cAMPS (Santa Cruz Biotechnology cat. # sc-215821); and TX-1123 (Santa Cruz Biotechnology cat. # sc-296675). Other FDA approved kinase inhibitors useful for the invention include, but are not limited to Gleevec

(Novartis), Herceptin (Genentech), Iressa (Astra-Zeneca), Erbitux (Imclone Systems), Tarceva (OSI Pharma), Nexavar (Bayer), Sutent (Pfizer), and Lapatinib (GSK), as well as those disclosed in Dar and Shokat (2011) Ann. Rev. Biochem. 80: 769-795, which is hereby incorporated by reference in its entirety.

[0083] Structures of DNA J-cAMP-dependent PK fusion molecule inhibitors useful for the invention include, but are not limited to:

-27-

; H-7, Rp-8-CPT-cAMPS,

; and TX-1123,

Assessment and Therapuetic Treatment

[0084] The invention provides a method of decreasing the growth of a solid tumor in a subject. The tumor is associated with, but not limited to, fibrolamellar hepatocellular carcinoma, hepatoblastoma, hepatocellular carcinoma, hepatocellular adenoma, and cavernous hemangioma. In one embodiment, the method comprises detecting the presence of a DNA J-cAMP-dependent PK fusion molecule in a sample obtained from a subject. In some embodiments, the sample is incubated with an agent that binds to a DNA J-cAMP-dependent PK fusion molecule, such as an antibody, a probe, a nucleic acid primer, and the like. In further embodiments, the method comprises administering to the subject an effective amount of a DNA J-cAMP-dependent PK fusion molecule inhibitor, wherein the inhibitor decreases the size of the solid tumor.

[0085] The invention also provides a method for treating or preventing a liver cancer in a subject. In one embodiment, the liver cancer is a pediatric liver cancer. In some embodiments, the liver cancer comprises fibrolamellar hepatocellular carcinoma, hepatoblastoma, hepatocellular carcinoma, hepatocellular adenoma, cavernous hemangioma, or a combination of the liver cancers described. In one embodiment, the method comprises detecting the presence of a DNA J-cAMP- dependent PK fusion molecule in a sample obtained from a subject, the presence of the fusion being indicative of a liver cancer, and, administering to the subject in need a therapeutic treatment against a liver cancer. In some embodiments, the sample is incubated with an agent that binds to a DNA J-cAMP-dependent PK fusion molecule, such as an antibody, a probe, a nucleic acid primer, and the like.

[0086] The invention also provides a method for decreasing in a subject in need thereof the expression level or activity of a fusion protein comprising a DNA J protein fused to a kinase domain of a cAMP-dependent protein kinase. In some embodiments, the method comprises obtaining a biological sample from the subject. In some embodiments, the sample is incubated with an agent that binds to a DNA J-cAMP-dependent PK fusion molecule, such as an antibody, a probe, a nucleic acid primer, and the like. In some embodiments, the method comprises administering to the subject a therapeutic amount of a composition comprising an admixture of a pharmaceutically acceptable carrier an inhibitor of the fusion protein of the invention. In another embodiment, the method further comprises determining the fusion protein expression level or activity. In another embodiment, the method further comprises detecting whether the fusion protein expression level or activity is decreased as compared to the fusion protein expression level or activity prior to administration of the composition, thereby decreasing the expression level or activity of the fusion protein. In some embodiments, the fusion protein is a DNA J-cAMP- dependent PK fusion fusion protein. In other embodiments, the fusion protein is a DNAJB1- PRKACA fusion protein.

[0087] The administering step in each of the claimed methods can comprise a drug

administration, such as DNA J-cAMP-dependent PK fusion molecule inhibitor (for example, a pharmaceutical composition comprising an antibody that specifically binds to a DNA J-cAMP- dependent PK fusion molecule or a fragment thereof (e.g., a DNAJB1-PRKACA fusion protein); a small molecule that specifically binds to a DNAJB1 protein; a small molecule that specifically binds to a PRKACA protein; an antisense RNA or antisense DNA that decreases expression of a DNA J-cAMP-dependent PK fusion molecule; a siRNA that specifically targets a gene encoding a DNA J-cAMP-dependent PK fusion molecule; or a combination thereof). In one embodiment, the therapeutic molecule to be administered comprises a polypeptide of a DNA J-cAMP-dependent PK fusion molecule, comprising at least about 75%, at least about 80%>, at least about 85%, at least about 90%), at least about 93%>, at least about 95%, at least about 97%, at least about 98%>, at least about 99%, or 100% of the amino acid sequence of SEQ ID NO: 1, and exhibits the function of decreasing expression of such a protein, thus treating a gene fusion-associated cancer. In another embodiment, administration of the therapeutic molecule decreases the size of the solid tumor associated with hepatoblastoma, hepatocellular carcinoma, hepatocellular adenoma, or cavernous hemangioma.

[0088] In another embodiment, the therapeutic molecule to be administered comprises an siRNA directed to a human nucleic acid sequence comprising a DNA J-cAMP-dependent PK fusion molecule. In one embodiment, the siRNA is directed to SEQ ID NO: 2. In a further embodiment, the therapeutic molecule to be administered comprises an antibody or binding fragment thereof, which is directed against SEQ ID NO: 1. In some embodiments, the therapeutic molecule to be administered comprises a small molecule that specifically binds to a PRKACA protein, such as 5- Iodotubercidin, A-3 Hydrochloride, KT 5720, ML-9, H-89, HA-1004, H7, A-674563, K252, or a combination thereof.

[0089] In one embodiment, the invention provides for the detection of a chromosomal rearrangement at given chromosomal coordinates. In another embodiment, the detection or determination comprises nucleic acid sequencing, selective hybridization, selective amplification, gene expression analysis, or a combination thereof. In another embodiment, the detection or determination comprises protein expression analysis, for example by western blot analysis, ELISA, or other antibody detection methods.

[0090] In one embodiment, the biological sample comprises liver cells, serum, bone marrow, blood, peripheral blood, lymph nodes, cerebro-spinal fluid, urine, a saliva sample, a buccal swab, a serum sample, a sputum sample, a lacrimal secretion sample, a semen sample, a vaginal secretion sample, a fetal tissue sample, or a combination thereof. [0091] A DNA J-cAMP-dependent PK fusion molecule, for example, a fusion between a J domain -containing protein and a kinase domain of a cAMP-dependent protein kinase (e.g., a DNAJBl-PRKACA fusion protein), can be determined at the level of the DNA, RNA, or polypeptide. Optionally, detection can be determined by performing an oligonucleotide ligation assay, a confirmation based assay, a hybridization assay, a sequencing assay, an allele-specific amplification assay, a microsequencing assay, a melting curve analysis, a denaturing high performance liquid chromatography (DHPLC) assay (for example, see Jones et al, (2000) Hum Genet., 106(6):663-8), or a combination thereof. In one embodiment, the detection is performed by sequencing all or part of a DNA J-cAMP-dependent PK fusion molecule (e.g., DNAJBl-PRKACA nucleic acid), or by selective hybridization or amplification of all or part of a DNA J-cAMP- dependent PK fusion molecule (e.g., a DNAJBl-PRKACA nucleic acid). A DNA J-cAMP- dependent PK fusion molecule specific amplification (e.g., a DNAJBl-PRKACA nucleic acid specific amplification) can be carried out before the fusion identification step.

[0092] The invention provides for a method of detecting a chromosomal alteration in a subject afflicted with a gene-fusion associated cancer. In one embodiment, the chromosomal alteration is an in-frame fused transcript described herein, for example a DNA J-cAMP-dependent PK fusion molecule. In some embodiments, the chromosomal alteration is a chromosomal translocation, for example a DNA J-cAMP-dependent PK fusion molecule. An alteration in a chromosome region occupied by a DNA J-cAMP-dependent PK fusion molecule, such as a DNAJBl-PRKACA nucleic acid, can be any form of mutation(s), deletion(s), rearrangement(s) and/or insertions in the coding and/or non-coding region of the locus, alone or in various combination(s). Mutations can include point mutations. Insertions can encompass the addition of one or several residues in a coding or non-coding portion of the gene locus. Insertions can comprise an addition of between 1 and 50 base pairs in the gene locus. Deletions can encompass any region of one, two or more residues in a coding or non-coding portion of the gene locus, such as from two residues up to the entire gene or locus. Deletions can affect smaller regions, such as domains (introns) or repeated sequences or fragments of less than about 50 consecutive base pairs, although larger deletions can occur as well. Rearrangement includes inversion of sequences. The alteration in a chromosome region occupied by a DNA J-cAMP-dependent PK fusion molecule, e.g., a DNAJBl-PRKACA nucleic acid, can result in amino acid substitutions, RNA splicing or processing, product instability, the creation of stop codons, production of oncogenic fusion proteins, frame-shift mutations, and/or truncated polypeptide production. The alteration can result in the production of a DNA J-cAMP-dependent PK fusion molecule, for example, one encoded by a DNAJBl-PRKACA nucleic acid, with altered function, stability, targeting or structure. The alteration can also cause a reduction, or even an increase in protein expression. In one embodiment, the alteration in the chromosome region occupied by a DNA J-cAMP-dependent PK fusion molecule can comprise a chromosomal rearrangement resulting in the production of a DNA J-cAMP-dependent PK fusion molecule, such as a DNAJBl-PRKACA fusion. This alteration can be determined at the level of the DNA, RNA, or polypeptide. In another embodiment, the detection or determination comprises nucleic acid sequencing, selective hybridization, selective amplification, gene expression analysis, or a combination thereof. In another embodiment, the detection or determination comprises protein expression analysis, for example by western blot analysis, ELISA, or other antibody detection methods. In one embodiment, the coordinates comprising the DNAJBl-PRKACA fusion combines exon 1 of DNAJB1 (chrl9: 14628951) and the beginning of exon2 of PRKACA (chrl9: 14218221).

[0093] The present invention provides a method for treating a gene-fusion associated cancer in a subject in need thereof. In one embodiment, the method comprises obtaining a sample from the subject to determine the level of expression of a DNA J-cAMP-dependent PK fusion molecule in the subject. In some embodiments, the sample is incubated with an agent that binds to a DNA J- cAMP-dependent PK fusion molecule, such as an antibody, a probe, a nucleic acid primer, and the like. In another embodiment, the detection or determination comprises nucleic acid sequencing, selective hybridization, selective amplification, gene expression analysis, or a combination thereof. In another embodiment, the detection or determination comprises protein expression analysis, for example by western blot analysis, ELISA, or other antibody detection methods. In some embodiments, the method further comprises assessing whether to administer a DNA J-cAMP- dependent PK fusion molecule inhibitor based on the expression pattern of the subject. In further embodiments, the method comprises administering a DNA J-cAMP-dependent PK fusion molecule inhibitor to the subject. In one embodiment, the cancer comprises liver cancer. In another embodiment, the liver cancer is a pediatric liver cancer. In some embodiments, the liver cancer comprises hepatoblastoma, hepatocellular carcinoma, hepatocellular adenoma, cavernous hemangioma, or a combination of the liver cancers described.

[0094] In one embodiment, the invention provides for a method of detecting the presence of altered RNA expression of a DNA J-cAMP-dependent PK fusion molecule in a subject, for example one afflicted with a gene-fusion associated cancer. In another embodiment, the invention provides for a method of detecting the presence of a DNA J-cAMP-dependent PK fusion molecule in a subject. In some embodiments, the method comprises obtaining a sample from the subject to determine whether the subject expresses a DNA J-cAMP-dependent PK fusion molecule. In some embodiments, the sample is incubated with an agent that binds to a DNA J-cAMP-dependent PK fusion molecule, such as an antibody, a probe, a nucleic acid primer, and the like. In other embodiments, the detection or determination comprises nucleic acid sequencing, selective hybridization, selective amplification, gene expression analysis, or a combination thereof. In another embodiment, the detection or determination comprises protein expression analysis, for example by western blot analysis, ELISA, or other antibody detection methods. In some embodiments, the method further comprises assessing whether to administer a DNA J-cAMP- dependent PK fusion molecule inhibitor based on the expression pattern of the subject. In further embodiments, the method comprises administering a DNA J-cAMP-dependent PK fusion molecule inhibitor to the subject. Altered RNA expression includes the presence of an altered RNA sequence, the presence of an altered RNA splicing or processing, or the presence of an altered quantity of RNA. These can be detected by various techniques known in the art, including sequencing all or part of the RNA or by selective hybridization or selective amplification of all or part of the RNA. In a further embodiment, the method can comprise detecting the presence or expression of a DNA J-cAMP-dependent PK fusion molecule, such as one encoded by a DNAJB 1- PRKACA nucleic acid. Altered polypeptide expression includes the presence of an altered polypeptide sequence, the presence of an altered quantity of polypeptide, or the presence of an altered tissue distribution. These can be detected by various techniques known in the art, including by sequencing and/or binding to specific ligands (such as antibodies). In one embodiment, the detecting comprises using a northern blot; real time PCR and primers directed to SEQ ID NO: 2; a ribonuclease protection assay; a hybridization, amplification, or sequencing technique to detect an DNA J-cAMP-dependent PK fusion molecule, such as one comprising SEQ ID NO: 2; or a combination thereof. In another embodiment, the PCR primers are directed to SEQ ID NO: 2. In a further embodiment, primers used for the screening of DNA J-cAMP-dependent PK fusion molecules, such as a DNAJB 1 -PRKAC A fusion, are directed to SEQ ID NO: 2.

[0095] Various techniques known in the art can be used to detect or quantify altered gene or RNA expression or nucleic acid sequences, which include, but are not limited to, hybridization, sequencing, amplification, and/or binding to specific ligands (such as antibodies). Other suitable methods include allele-specific oligonucleotide (ASO), oligonucleotide ligation, allele-specific amplification, Southern blot (for DNAs), Northern blot (for RNAs), single-stranded conformation analysis (SSCA), PFGE, fluorescent in situ hybridization (FISH), gel migration, clamped denaturing gel electrophoresis, denaturing HLPC, melting curve analysis, heteroduplex analysis, RNase protection, chemical or enzymatic mismatch cleavage, ELISA, radio-immunoassays (RIA) and immuno-enzymatic assays (IEMA). [0096] Some of these approaches (such as SSCA and constant gradient gel electrophoresis (CGGE)) are based on a change in electrophoretic mobility of the nucleic acids, as a result of the presence of an altered sequence. According to these techniques, the altered sequence is visualized by a shift in mobility on gels. The fragments can then be sequenced to confirm the alteration. Some other approaches are based on specific hybridization between nucleic acids from the subject and a probe specific for wild type or altered gene or RNA. The probe can be in suspension or

immobilized on a substrate. The probe can be labeled to facilitate detection of hybrids. Some of these approaches are suited for assessing a polypeptide sequence or expression level, such as Northern blot, ELISA and RIA. These latter require the use of a ligand specific for the polypeptide, for example, the use of a specific antibody.

[0097] Hybridization. Hybridization detection methods are based on the formation of specific hybrids between complementary nucleic acid sequences that serve to detect nucleic acid sequence alteration(s). A detection technique involves the use of a nucleic acid probe specific for a wild type or altered gene or RNA, followed by the detection of the presence of a hybrid. The probe can be in suspension or immobilized on a substrate or support (for example, as in nucleic acid array or chips technologies). The probe can be labeled to facilitate detection of hybrids. In one embodiment, the probe according to the invention can comprise a nucleic acid directed to SEQ ID NO: 2. For example, a sample from the subject can be contacted with a nucleic acid probe specific for a gene encoding a DNA J-cAMP-dependent PK fusion molecule, and the formation of a hybrid can be subsequently assessed. In one embodiment, the method comprises contacting simultaneously the sample with a set of probes that are specific for a DNA J-cAMP-dependent PK fusion molecule. Also, various samples from various subjects can be investigated in parallel.

[0098] According to the invention, a probe can be a polynucleotide sequence which is complementary to and specifically hybridizes with a, or a target portion of a, gene or RNA corresponding to a DNA J-cAMP-dependent PK fusion molecule. Useful probes are those that are complementary to the gene, RNA, or target portion thereof. Probes can comprise single-stranded nucleic acids of between 8 to 1000 nucleotides in length, for instance between 10 and 800, between 15 and 700, or between 20 and 500. Longer probes can be used as well. A useful probe of the invention is a single stranded nucleic acid molecule of between 8 to 500 nucleotides in length, which can specifically hybridize to a region of a gene or RNA that corresponds to a DNA J-cAMP- dependent PK fusion molecule.

[0099] The sequence of the probes can be derived from the sequences of the DNA J-cAMP- dependent PK fusion genes provided herein. Nucleotide substitutions can be performed, as well as chemical modifications of the probe. Such chemical modifications can be accomplished to increase the stability of hybrids (e.g., intercalating groups) or to label the probe. Some examples of labels include, without limitation, radioactivity, fluorescence, luminescence, and enzymatic labeling.

[00100] A guide to the hybridization of nucleic acids is found in e.g., Sambrook, ed., Molecular Cloning: A Laboratory Manual (3^rd Ed.), Vols. 1-3, Cold Spring Harbor Laboratory, 1989; Current Protocols In Molecular Biology, Ausubel, ed. John Wiley & Sons, Inc., New York, 2001;

Laboratory Techniques In Biochemistry And Molecular Biology: Hybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y., 1993.

[00101] Sequencing. Sequencing can be carried out using techniques well known in the art, using automatic sequencers. The sequencing can be performed on the complete DNA J-cAMP- dependent PK fusion molecule or on specific domains thereof.

[00102] Amplification. Amplification is based on the formation of specific hybrids between complementary nucleic acid sequences that serve to initiate nucleic acid reproduction.

Amplification can be performed according to various techniques known in the art, such as by polymerase chain reaction (PCR), ligase chain reaction (LCR), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA). These techniques can be performed using commercially available reagents and protocols. Useful techniques in the art encompass real-time PCR, allele-specific PCR, or PCR based single-strand conformational polymorphism (SSCP). Amplification usually requires the use of specific nucleic acid primers, to initiate the reaction. For example, nucleic acid primers useful for amplifying sequences corresponding to a DNA J-cAMP-dependent PK fusion molecule are able to specifically hybridize with a portion of the gene locus that flanks a target region of the locus. In one embodiment, amplification comprises using forward and reverse PCR primers directed to SEQ ID NO: 2.

Nucleic acid primers useful for amplifying sequences from a DNA J-cAMP-dependent PK fusion molecule (e.g., a DNAJBl-PRKACA nucleic acid); the primers specifically hybridize with a portion of a DNA J-cAMP-dependent PK fusion molecule. In certain subjects, the presence of a DNA J-cAMP-dependent PK fusion molecule corresponds to a subject with a liver cancer. In one embodiment, amplification can comprise using forward and reverse PCR primers directed to SEQ ID NO: 2.

[00103] Non-limiting amplification methods include, e.g., polymerase chain reaction, PCR (PCR Protocols, A Guide To Methods And Applications, ed. Innis, Academic Press, N.Y., 1990 and PCR Strategies, 1995, ed. Innis, Academic Press, Inc., N.Y.); ligase chain reaction (LCR) (Wu (1989) Genomics 4:560; Landegren (1988) Science 241 : 1077; Barringer (1990) Gene 89: 117); transcription amplification (Kwoh (1989) PNAS 86: 1173); and, self-sustained sequence replication (Guatelli (1990) PNAS 87: 1874); Q Beta replicase amplification (Smith (1997) J. Clin. Microbiol. 35: 1477-1491), automated Q-beta replicase amplification assay (Burg (1996) Mol. Cell. Probes 10:257-271) and other R A polymerase mediated techniques (e.g., NASBA, Cangene,

Mississauga, Ontario; see also Berger (1987) Methods Enzymol. 152:307-316; U.S. Pat. Nos.

4,683,195 and 4,683,202; and Sooknanan (1995) Biotechnology 13:563-564). All the references stated above are incorporated by reference in their entireties.

[00104] The invention provides for a nucleic acid primer, wherein the primer can be

complementary to and hybridize specifically to a portion of a DNA J-cAMP-dependent PK fusion molecule, such as a DNAJBl-PRKACA nucleic acid (e.g., DNA or RNA) in certain subjects having a gene fusion-associated cancer. In one embodiment, the cancer comprises liver cancer. In another embodiment, the liver cancer is a pediatric liver cancer. In some embodiments, the liver cancer comprises hepatoblastoma, hepatocellular carcinoma, hepatocellular adenoma, cavernous hemangioma, or a combination of the liver cancers described. Primers of the invention can be specific for fusion sequences of a DNAJBl-PRKACA nucleic acid (DNA or RNA). By using such primers, the detection of an amplification product indicates the presence of a fusion of a DNAJBl- PRKACA nucleic acid. Examples of primers of this invention can be single-stranded nucleic acid molecules of about 5 to 60 nucleotides in length, or about 8 to about 25 nucleotides in length. The sequence can be derived directly from the sequence of a DNA J-cAMP-dependent PK fusion molecule, e.g. a DNAJBl-PRKACA nucleic acid. Perfect complementarity is useful to ensure high specificity; however, certain mismatch can be tolerated. For example, a nucleic acid primer or a pair of nucleic acid primers as described above can be used in a method for detecting the presence of a liver cancer in a subject. In one embodiment, primers can be used to detect a DNA J-cAMP- dependent PK fusion molecule, such as a primer directed to SEQ ID NO: 2; or a combination thereof.

[00105] Specific Ligand Binding. As discussed herein, a nucleic acid encoding a DNA J-cAMP- dependent PK fusion molecule or expression of a DNA J-cAMP-dependent PK fusion molecule, can also be detected by screening for alteration(s) in a sequence or expression level of a

polypeptide encoded by the same. Different types of ligands can be used, such as specific antibodies. In one embodiment, the sample is contacted with an antibody specific for a polypeptide encoded by a DNA J-cAMP-dependent PK fusion molecule and the formation of an immune complex is subsequently determined. Various methods for detecting an immune complex can be used, such as ELISA, radioimmunoassays (RIA) and immuno-enzymatic assays (IEMA).

[00106] For example, an antibody can be a polyclonal antibody, a monoclonal antibody, as well as fragments or derivatives thereof having substantially the same antigen specificity. Fragments include Fab, Fab'2, or CDR regions. Derivatives include single-chain antibodies, humanized antibodies, or poly-functional antibodies. An antibody specific for a polypeptide encoded by a DNA J-cAMP-dependent PK fusion molecule can be an antibody that selectively binds such a polypeptide. In one embodiment, the antibody is raised against a polypeptide encoded by a DNA J- cAMP-dependent PK fusion molecule (such as a DNAJBl-PRKACA fusion) or an epitope- containing fragment thereof. Although non-specific binding towards other antigens can occur, binding to the target polypeptide occurs with a higher affinity and can be reliably discriminated from non-specific binding. In one embodiment, the method can comprise contacting a sample from the subject with an antibody specific for a DNA J-cAMP-dependent PK fusion molecule, and determining the presence of an immune complex. Optionally, the sample can be contacted to a support coated with antibody specific for a DNA J-cAMP-dependent PK fusion molecule. In one embodiment, the sample can be contacted simultaneously, or in parallel, or sequentially, with various antibodies specific for different forms of a DNA J-cAMP-dependent PK fusion molecule, e.g., a DNAJBl-PRKACA fusion.

[00107] The invention also provides for a diagnostic kit comprising products and reagents for detecting in a sample from a subject the presence of a DNA J-cAMP-dependent PK fusion molecule. The kit can be useful for determining whether a sample from a subject exhibits increased or reduced expression of a DNA J-cAMP-dependent PK fusion molecule. For example, the diagnostic kit according to the present invention comprises any primer, any pair of primers, any nucleic acid probe and/or any ligand, or any antibody directed specifically to a DNA J-cAMP- dependent PK fusion molecule. The diagnostic kit according to the present invention can further comprise reagents and/or protocols for performing a hybridization, amplification, or antigen- antibody immune reaction. In one embodiment, the kit can comprise nucleic acid primers that specifically hybridize to and can prime a polymerase reaction from a DNA J-cAMP-dependent PK fusion molecule directed to SEQ ID NO: 2, or a combination thereof. In one embodiment, primers can be used to detect a DNA J-cAMP-dependent PK fusion molecule, such as a primer directed to SEQ ID NO: 2. In a further embodiment, primers used for the screening of DNA J-cAMP- dependent PK fusion molecules, such as DNAJBl-PRKACA fusions, are directed to SEQ ID NO: 2. In some embodiments, primers used for genomic detection of a DNAJBl-PRKACA fusion are directed to SEQ ID NO: 2. In some embodiments, the kit comprises an antibody that specifically binds to a DNA J-cAMP-dependent PK fusion molecule comprising SEQ ID NO: 1, wherein the antibody will recognize the protein only when a DNA J-cAMP-dependent PK fusion molecule is present

[00108] The diagnosis methods can be performed in vitro, ex vivo, or in vivo. These methods utilize a sample from the subject in order to assess the status of a DNA J-cAMP-dependent PK fusion molecule. The sample can be any biological sample derived from a subject, which contains nucleic acids or polypeptides. Examples of such samples include, but are not limited to, fluids, tissues, cell samples, organs, and tissue biopsies. Non-limiting examples of samples include blood, liver, plasma, serum, saliva, urine, or seminal fluid. The sample can be collected according to conventional techniques and used directly for diagnosis or stored. The sample can be treated prior to performing the method, in order to render or improve availability of nucleic acids or

polypeptides for testing. Treatments include, for instance, lysis (e.g., mechanical, physical, or chemical), centrifugation. The nucleic acids and/or polypeptides can be pre -purified or enriched by conventional techniques, and/or reduced in complexity. Nucleic acids and polypeptides can also be treated with enzymes or other chemical or physical treatments to produce fragments thereof. In one embodiment, the sample is contacted with reagents, such as probes, primers, or ligands, in order to assess the presence of a DNA J-cAMP-dependent PK fusion molecule. Contacting can be performed in any suitable device, such as a plate, tube, well, or glass. In some embodiments, the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array. The substrate can be a solid or semi-solid substrate such as any support comprising glass, plastic, nylon, paper, metal, or polymers. The substrate can be of various forms and sizes, such as a slide, a membrane, a bead, a column, or a gel. The contacting can be made under any condition suitable for a complex to be formed between the reagent and the nucleic acids or polypeptides of the sample.

Nucleic Acid Delivery Methods

[00109] Delivery of nucleic acids into viable cells can be effected ex vivo, in situ, or in vivo by use of vectors, such as viral vectors (e.g., lentivirus, adenovirus, adeno-associated virus, or a retrovirus), or ex vivo by use of physical DNA transfer methods (e.g., liposomes or chemical treatments). Non-limiting techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, and the calcium phosphate precipitation method (See, for example, Anderson, Nature, 1998) supplement to 392(6679):25(). Introduction of a nucleic acid or a gene encoding a polypeptide of the invention can also be accomplished with extrachromosomal substrates (transient expression) or artificial chromosomes (stable expression). Cells can also be cultured ex vivo in the presence of therapeutic compositions of the present invention in order to proliferate or to produce a desired effect on or activity in such cells. Treated cells can then be introduced in vivo for therapeutic purposes.

[00110] Nucleic acids can be inserted into vectors and used as gene therapy vectors. A number of viruses have been used as gene transfer vectors, including papovaviruses, e.g., SV40 (Madzak et al, (1992) J Gen Virol. 73( Pt 6): 1533-6), adenovirus (Berkner (1992) Curr Top Microbiol ImmunolA5% 39-66; Berkner (1988) Biotechniques, 6(7):616-29; Gorziglia and Kapikian (1992) J Virol. 66(7):4407-12; Quantin et al, (1992) Proc Natl Acad Sci USA. 89(7):2581-4; Rosenfeld et al, (1992) Cell. 68(1): 143-55; Wilkinson et al, (1992) Nucleic Acids Res. 20(9):2233-9; Stratford- Perricaudet et al, (1990) Hum Gene Ther. l(3):241-56), vaccinia virus (Moss (1992) Curr Opin Biotechnol. 3(5):518-22), adeno-associated virus (Muzyczka, (1992) Curr Top Microbiol Immunol . 158:97-129; Ohi et al, (1990) Gene. 89(2):279-82), herpesviruses including HSV and EBV

(Margolskee (1992) Curr Top Microbiol Immunol. 158:67-95; Johnson et al, (1992) Brain Res Mol Brain Res.l2(l-3):95-102; Fink et al, (1992) Hum Gene Ther. 3(1):11-9; Breakefield and Geller (\9 l) Mol Neurobiol. 1(4):339-71; Freese et al, (1990) Biochem Pharmacol. 40(10):2189-99), and retroviruses of avian (Bandyopadhyay and Temin (1984) Mol Cell Biol. 4(4):749-54;

Petropoulos et al, (1992) J Virol. 66(6):3391-7), murine (Miller et al. (1992) Mol Cell Biol.

12(7):3262-72; Miller et al, (1985) J Virol. 55(3):521-6; Sorge et al, (1984) Mol Cell Biol.

4(9): 1730-7; Mann and Baltimore (1985) J Virol. 54(2):401-7; Miller et al, (1988) J Virol.

62(11):4337-45), and human origin (Shimada et al, (1991) J Clin Invest. 88(3): 1043-7; Helseth et al, (1990) J Virol. 64(12):6314-8; Page et al, (1990) J Virol. 64(11):5270-6; Buchschacher and Panganiban (1992) J Virol. 66(5):2731-9).

[00111] Non- limiting examples of in vivo gene transfer techniques include trans fection with viral (e.g., retroviral) vectors (see U.S. Pat. No. 5,252,479, which is incorporated by reference in its entirety) and viral coat protein-liposome mediated transfection (Dzau et al., (1993) Trends in Biotechnology 11 :205-210), incorporated entirely by reference). For example, naked DNA vaccines are generally known in the art; see Brower, (1998) Nature Biotechnology, 16: 1304-1305, which is incorporated by reference in its entirety. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Pat. No.

5,328,470) or by stereotactic injection (see, e.g., Chen, et al, (1994) Proc. Natl. Acad. Sci. USA 91 :3054-3057). The pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

[00112] For reviews of nucleic acid delivery protocols and methods see Anderson et al. (1992) Science 256:808-813; U.S. Pat. Nos. 5,252,479, 5,747,469, 6,017,524, 6,143,290, 6,410,010 6,511,847; and U.S. Application Publication No. 2002/0077313, which are all hereby incorporated by reference in their entireties. For additional reviews, see Friedmann (1989) Science, 244: 1275- 1281; Verma, Scientific American: 68-84 (1990); Miller (1992) Nature, 357: 455-460; Kikuchi et al. (2008) J Dermatol Sci. 50(2):87-98; Isaka et al. (2007) Expert Opin Drug Deliv. 4(5):561-71; Jager et al.(2007) Curr Gene Ther. 7(4):272-83; Waehler et al.(2007) Nat Rev Genet. 8(8):573-87; Jensen et al. (2007) Ann Med. 39(2): 108-15; Herweijer et al. (2007) Gene Ther. 14(2):99-107; Eliyahu et al. (2005) Molecules 10(l):34-64; and Altaras et al. (2005) Adv Biochem Eng

Biotechnol. 99: 193-260, all of which are hereby incorporated by reference in their entireties.

[00113] A DNA J-cAMP-dependent PK fusion nucleic acid can also be delivered in a controlled release system. For example, the DNA J-cAMP-dependent PK fusion molecule can be

administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump can be used (see Sefton (1987) Biomed. Eng. 14:201; Buchwald et al. (1980) Surgery 88:507; Saudek et al. (1989) N. Engl. J. Med. 321 :574). In another embodiment, polymeric materials can be used (see Medical

Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, (1983) J. Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al. (1985) Science 228: 190; During et al. (1989) Ann. Neurol. 25:351;

Howard et al. (1989) J. Neurosurg. 71 : 105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems are discussed in the review by Langer (Science (1990) 249: 1527-1533).

Pharmaceutical Compositions and Administration for Therapy

[00114] An inhibitor of the invention can be incorporated into pharmaceutical compositions suitable for administration, for example the inhibitor and a pharmaceutically acceptable carrier [00115] A DNA J-cAMP-dependent PK fusion molecule or inhibitor of the invention can be administered to the subject once (e.g., as a single injection or deposition). Alternatively, a DNA J- cAMP-dependent PK fusion molecule or inhibitor can be administered once or twice daily to a subject in need thereof for a period of from about two to about twenty-eight days, or from about seven to about ten days. A DNA J-cAMP-dependent PK fusion molecule or inhibitor can also be administered once or twice daily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof. Furthermore, a DNA J-cAMP-dependent PK fusion molecule or inhibitor of the invention can be co-administrated with another therapeutic. Where a dosage regimen comprises multiple administrations, the effective amount of the DNA J-cAMP-dependent PK fusion molecule or inhibitor administered to the subject can comprise the total amount of gene product administered over the entire dosage regimen.

[00116] A DNA J-cAMP-dependent PK fusion molecule or inhibitor can be administered to a subject by any means suitable for delivering the DNA J-cAMP-dependent PK fusion molecule or inhibitor to cells of the subject, such as liver cancer cells, e.g., hepatoblastoma, hepatocellular carcinoma, hepatocellular adenoma, cavernous hemangioma, or a combination of the liver cancers described. For example, a DNA J-cAMP-dependent PK fusion molecule or inhibitor can be administered by methods suitable to transfect cells. Transfection methods for eukaryotic cells are well known in the art, and include direct injection of the nucleic acid into the nucleus or pronucleus of a cell; electroporation; liposome transfer or transfer mediated by lipophilic materials; receptor mediated nucleic acid delivery, bioballistic or particle acceleration; calcium phosphate

precipitation, and transfection mediated by viral vectors.

[00117] The compositions of this invention can be formulated and administered to reduce the symptoms associated with a liver cancer, e.g., hepatoblastoma, hepatocellular carcinoma, hepatocellular adenoma, cavernous hemangioma, or a combination of the liver cancers described, by any means that produces contact of the active ingredient with the agent's site of action in the body of a subject, such as a human or animal (e.g., a dog, cat, or horse). They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic active ingredients or in a combination of therapeutic active ingredients. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

[00118] A therapeutically effective dose of DNA J-cAMP-dependent PK fusion molecule or inhibitor can depend upon a number of factors known to those or ordinary skill in the art. The dose(s) of the DNA J-cAMP-dependent PK fusion molecule inhibitor can vary, for example, depending upon the identity, size, and condition of the subject or sample being treated, further depending upon the route by which the composition is to be administered, if applicable, and the effect which the practitioner desires the a DNA J-cAMP-dependent PK fusion molecule inhibitor to have upon the nucleic acid or polypeptide of the invention. These amounts can be readily determined by a skilled artisan. Any of the therapeutic applications described herein can be applied to any subject in need of such therapy, including, for example, a mammal such as a dog, a cat, a cow, a horse, a rabbit, a monkey, a pig, a sheep, a goat, or a human.

[00119] Pharmaceutical compositions for use in accordance with the invention can be

formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The therapeutic compositions of the invention can be formulated for a variety of routes of administration, including systemic and topical or localized administration. Techniques and formulations generally can be found in Remmington's Pharmaceutical Sciences, Meade Publishing Co., Easton, Pa (20^th Ed., 2000), the entire disclosure of which is herein incorporated by reference. For systemic administration, an injection is useful, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the therapeutic compositions of the invention can be formulated in liquid solutions, for example in physiologically compatible buffers such as Hank's solution or Ringer's solution. In addition, the therapeutic compositions can be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included. Pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen-free. These pharmaceutical formulations include formulations for human and veterinary use.

[00120] According to the invention, a pharmaceutically acceptable carrier can comprise any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Any

conventional media or agent that is compatible with the active compound can be used.

Supplementary active compounds can also be incorporated into the compositions.

[00121] A pharmaceutical composition containing DNA J-cAMP-dependent PK fusion molecule inhibitor can be administered in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed herein. Such pharmaceutical compositions can comprise, for example antibodies directed to a DNA J-cAMP-dependent PK fusion molecule, or a variant thereof, or antagonists of a DNA J-cAMP-dependent PK fusion molecule. The compositions can be administered alone or in combination with at least one other agent, such as a stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water. The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones.

[00122] Sterile injectable solutions can be prepared by incorporating the DNA J-cAMP- dependent PK fusion molecule inhibitor (e.g., a polypeptide or antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze- drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[00123] In some embodiments, the DNA J-cAMP-dependent PK fusion molecule inhibitor can be applied via transdermal delivery systems, which slowly releases the active compound for percutaneous absorption. Permeation enhancers can be used to facilitate transdermal penetration of the active factors in the conditioned media. Transdermal patches are described in for example, U.S. Pat. No. 5,407,713; U.S. Pat. No. 5,352,456; U.S. Pat. No. 5,332,213; U.S. Pat. No. 5,336,168; U.S. Pat. No. 5,290,561; U.S. Pat. No. 5,254,346; U.S. Pat. No. 5,164,189; U.S. Pat. No. 5,163,899; U.S. Pat. No. 5,088,977; U.S. Pat. No. 5,087,240; U.S. Pat. No. 5,008,110; and U.S. Pat. No. 4,921,475.

[00124] "Subcutaneous" administration can refer to administration just beneath the skin (i.e., beneath the dermis). Generally, the subcutaneous tissue is a layer of fat and connective tissue that houses larger blood vessels and nerves. The size of this layer varies throughout the body and from person to person. The interface between the subcutaneous and muscle layers can be encompassed by subcutaneous administration. This mode of administration can be feasible where the

subcutaneous layer is sufficiently thin so that the factors present in the compositions can migrate or diffuse from the locus of administration. Thus, where intradermal administration is utilized, the bolus of composition administered is localized proximate to the subcutaneous layer.

[00125] Administration of cell aggregates is not restricted to a single route, but can encompass administration by multiple routes. For instance, exemplary administrations by multiple routes include, among others, a combination of intradermal and intramuscular administration, or intradermal and subcutaneous administration. Multiple administrations can be sequential or concurrent. Other modes of application by multiple routes will be apparent to the skilled artisan. [00126] In other embodiments, this implantation method will be a one-time treatment for some subjects. In further embodiments of the invention, multiple cell therapy implantations will be required. In some embodiments, the cells used for implantation will generally be subject- specific genetically engineered cells. In another embodiment, cells obtained from a different species or another individual of the same species can be used. Thus, using such cells can require

administering an immunosuppressant to prevent rejection of the implanted cells. Such methods have also been described in United States Patent No. 7,419,66 land PCT application publication WO 2001/32840, and are hereby incorporated by reference.

[00127] A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation or ingestion), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediammetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[00128] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[00129] Sterile injectable solutions can be prepared by incorporating the inhibitor (e.g., a polypeptide or antibody or small molecule) of the invention in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation of sterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze- drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[00130] Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier and subsequently swallowed.

[00131] Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: 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 or sterotes; 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.

[00132] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.

Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. [00133] In some embodiments, the effective amount of the administered DNA J-cAMP- dependent PK fusion molecule inhibitor is at least about 0.0001 μ§/1¾ body weight, at least about 0.00025 μ§/1¾ body weight, at least about 0.0005 μg/kg body weight, at least about 0.00075 μg/kg body weight, at least about 0.001 μg/kg body weight, at least about 0.0025 μg/kg body weight, at least about 0.005 μg/kg body weight, at least about 0.0075 μg/kg body weight, at least about 0.01 μg/kg body weight, at least about 0.025 μg/kg body weight, at least about 0.05 μg/kg body weight, at least about 0.075 μg/kg body weight, at least about 0.1 μg/kg body weight, at least about 0.25 μg/kg body weight, at least about 0.5 μg/kg body weight, at least about 0.75 μg/kg body weight, at least about 1 μg/kg body weight, at least about 5 μg/kg body weight, at least about 10 μg/kg body weight, at least about 25 μg/kg body weight, at least about 50 μg/kg body weight, at least about 75 μg/kg body weight, at least about 100 μg/kg body weight, at least about 150 μg/kg body weight, at least about 200 μg/kg body weight, at least about 250 μg/kg body weight, at least about 300 μg/kg body weight, at least about 350 μg/kg body weight, at least about 400 μg/kg body weight, at least about 450 μg/kg body weight, at least about 500 μg/kg body weight, at least about 550 μg/kg body weight, at least about 600 μg/kg body weight, at least about 650 μg/kg body weight, at least about 700 μg/kg body weight, at least about 750 μg/kg body weight, at least about 800 μg/kg body weight, at least about 850 μg/kg body weight, at least about 900 μg/kg body weight, at least about 950 μg/kg body weight, at least about 1,000 μg/kg body weight, at least about 2,000 μg/kg body weight, at least about 3,000 μg/kg body weight, at least about 4,000 μg/kg body weight, at least about 5,000 μg/kg body weight, at least about 6,000 μg/kg body weight, at least about 7,000 μg/kg body weight, at least about 8,000 μg/kg body weight, at least about 9,500 μg/kg body weight, or at least about 10,000 μg/kg body weight.

* * *

[00134] 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. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[00135] All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated by reference. Publications and references cited herein are not admitted to be prior art. EXAMPLES

[00136] Examples are provided below to facilitate a more complete understanding of the invention. The following examples illustrate the exemplary modes of making and practicing the invention. However, the scope of the invention is not limited to specific embodiments disclosed in these Examples, which are for purposes of illustration only, since alternative methods can be utilized to obtain similar results.

[00137] Example 1: DNAJB 1 PRKACA fusion gene

[00138] Transcriptome sequencing (stranded total RNA with ribo-depletion) was performed from 11 patients: 9 normal and 20 tumor (primary and metastases) tissue samples. To identify fusion genes, several programs were used, including Tophat-fusion, Chimerascan and

FusionCatcher (Kangaspeska, S., Hultsch, S., Edgren, H., Nicorici, D., Murumagi, A., &

Kallioniemi, O. (2012). Reanalysis of RNA-sequencing data reveals several additional fusion genes with multiple isoforms. PLoS ONE, 7(10), e48745). Tophat-fusion and Chimerascan returned between 170,000-250,000 and 6,000-13,000 candidate fusions per sample, respectively (Kim and Salzberg (2011) Genome Biol. 12(8):R72; Carrara et al. (2013) Biomed Res Int. 2013:340620; Iyer et al (2011) Bioinformatics. 27(20):2903-4). To generate a more conservative set of candidates, FusionCatcher (version 0.99, http://code.google.eom/p/fusioncatcher/) was also run, which imposes a stringent set of filters on putative fusion transcripts on all samples (3 tumor samples are still in processing). FusionCatcher reported between 3 and 13 candidate fusions per sample. The candidate fusions were annotated to filter out false positives (known fusions, paralogs, read through between 2 adjacents genes, fusion reported in a "black list" of common false positives).

[00139] The fusion DNAJB 1 -PRKACA was reported in the 17 tumors samples and in none of the normal samples. There was no indication of false positive annotation for this fusion. The fusion transcript is supported by 10-280 reads per sample (mean 130 reads). At the transcript level, the fusion combines exon 1 of DNAJB 1 (chrl 9: 14628951 on reference sequence hgl9) and the beginning of exon2 of PRKACA (chrl 9: 14218221 on reference sequence hgl9).

[00140] Both genes are on the reverse strand. The fusion does not induce a frameshift. The fusion transcript retains the full catalytic domain of PRKACA as annotated in pFAM.

[00141] PRKACA is consistently over-expressed in tumor versus normal samples (between 2 and 25-fold). [00142] In addition, whole genome sequencing was performed of 10 patients (tumor/normal pairs at 60x/30x) and analyzed the mapped reads (BWA alignment, GATK realignment, GATK base recalibration (Li and Durbin (2010) Bioinformatics. 26(5):589-95; Li and Durbin (2009) Bioinformatics. 25(14):1754-60; McKenna et al. (2010) Genome Res. 20: 1297-303; DePristo et al. (2011) Nature Genetics. 43:491-498.)) with three structural variant (SV) detection tools:

BreakDancer, Delly, and BIC-Seq. BreakDancer and Delly locate discordant paired-end reads to predict SVs, while BIC-Seq compares read-depth ratios between tumor and normal. The joint output of all these programs is being analyzed to identify common structural variants. However, a focused look has been taken at one particular genomic region to look for evidence at the DNA level of the fusion gene discovered in the RNA data. For all 10 patients, a large somatic deletion

(ranging from 401,551 to 409,26 lbp in size) was observed with breakpoints joining PRKACA (intronic sequence between exon 1 and 2) with DNAJB1 (intronic sequence between exonl and 2 in 7 of the 10 cases and exon 2 in 3 cases). For two patients, the deletion is predicted by all three tools; for three patients it is identified by two of the tools, and for the remaining five patients it is identified by one of the tools. After manual inspection, we identified supporting read pairs for all but one tumor (which however had a clear read depth drop identified by BIC-seq), but no evidence for the deletion in the normal samples.

[00143] The table below shows the coordinates of the deletions as found in the DNA. The columns represent the chromosome, start position, and end position. The deletions were discovered using the three tools BreakDancer[l], Delly[2] and BIC-Seq[3]. To locate the exact break points, SplazerS was used [4]. All analysis was done after preprocessing with BWA (read mapping), Picard (duplicate removal) and GATK (realignment, recalibration).

#chr start end

19 1422 6134 14 627685

19 14218306 14 627567

19 14225111 14 628041

19 1422 6300 14 627922

19 14224739 14 627612

19 14225573 14 628324

19 14220907 14 6281 61

19 14220501 14 628075

19 14220010 14 627681

19 142217 90 14 628632

[00144] References

[1] Chen, Ken, et al. "BreakDancer: an algorithm for high-resolution mapping of genomic structural variation." Nature Methods 6.9 (2009): 677-681. [2] Rausch, Tobias, et al. "DELLY: structural variant discovery by integrated paired-end and split- read analysis." Bioinformatics 28.18 (2012): i333-i339.

[3] Xi, Ruibin, et al. "Copy number variation detection in whole-genome sequencing data using the Bayesian information criterion." Proceedings of the National Academy of Sciences 108.46 (2011): E1128-E1136.

[4] Emde, Anne-Katrin, et al. "Detecting genomic indel variants with exact breakpoints in single- and paired-end sequencing data using SplazerS." Bioinformatics 28.5 (2012): 619-627.

Claims

What is claimed is:

1. A purified fusion protein comprising a heat shock protein fused to a kinase domain of a cAMP-dependent protein kinase.

2. A purified fusion protein comprising a DNA J protein fused to a kinase domain of a cAMP- dependent protein kinase.

3. A purified fusion protein comprising a DNAJB1 protein fused to a kinase domain of a

cAMP-dependent protein kinase.

4. A purified fusion protein comprising a DNAJB1 protein fused to a cAMP-dependent protein kinase catalytic subunit alpha protein.

5. The purified fusion protein of claim 1, wherein the heat shock protein is a DNA J protein.

6. The purified fusion protein of claim 2, wherein the DNA J protein is a DNAJB1 protein.

7. The purified fusion protein of claim 1, 2, or 3, wherein the cAMP-dependent protein kinase is cAMP-dependent protein kinase catalytic subunit alpha protein (PRKACA).

8. A purified fusion protein encoded by an DNAJB1 -PRKACA nucleic acid, wherein

DNAJB1 -PRKACA comprises exon 1 of DNAJB1 located on human chromosome 19 spliced 5' to a combination of exons 2-10 of PRKACA located on human chromosome 19.

9. A synthetic nucleic acid encoding the fusion protein of claim 1, 2, 3, 4, or 8.

10. A purified DNAJB 1 -PRKACA fusion protein comprising SEQ ID NO : 1.

11. A synthetic nucleic acid encoding a DNAJB 1 -PRKACA fusion protein, wherein the nucleic acid comprises SEQ ID NO: 2.

12. An antibody or antigen-binding fragment thereof, that specifically binds to a purified fusion protein comprising a DNA J protein fused to a kinase domain of a cAMP-dependent protein kinase.

13. The antibody or antigen-binding fragment of claim 12, wherein the DNA J protein is a DNAJB1 protein.

14. The antibody or antigen-binding fragment of claim 12, wherein the cAMP-dependent

protein kinase is PRKACA.

15. The antibody or antigen-binding fragment of claim 12, wherein the fusion protein is a

DNAJBl-PRKACA fusion protein.

16. The antibody or antigen-binding fragment of claim 15, wherein the DNAJBl-PRKACA fusion protein comprises the amino acid sequence of SEQ ID NO: 1.

17. A composition for decreasing in a subject the expression level or activity of a fusion protein comprising a DNA J protein fused to a kinase domain of a cAMP-dependent protein kinase, the composition in an admixture of a pharmaceutically acceptable carrier comprising an inhibitor of the fusion protein.

18. The composition of claim 17, wherein the fusion protein is DNAJBl-PRKACA fusion

protein.

19. The composition of claim 17, wherein the inhibitor comprises an antibody that specifically binds to a DNAJBl-PRKACA fusion protein or a fragment thereof; a small molecule that specifically binds to a DNAJB 1 protein; a small molecule that specifically binds to a PRKACA protein; an antisense RNA or antisense DNA that decreases expression of a DNAJBl-PRKACA fusion polypeptide; a siRNA that specifically targets a DNAJBl- PRKACA fusion gene; or a combination thereof.

20. The composition of claim 19, wherein the small molecule that specifically binds to a PRKACA protein comprises 5-Iodotubercidin, A-3 Hydrochloride, KT 5720, ML-9, H-89, HA-1004, H7, A-674563, K252, or a combination thereof.

21. A method for decreasing in a subject in need thereof the expression level or activity of a fusion protein comprising a DNA J protein fused to a kinase domain of a cAMP-dependent protein kinase, the method comprising:

(a) administering to the subject a therapeutic amount of a composition of claim 17; and

(b) determining whether the fusion protein expression level or activity is decreased compared to fusion protein expression level or activity prior to administration of the composition, thereby decreasing the expression level or activity of the fusion protein.

22. A method for treating a liver cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of a DNA J-cAMP-dependent PK fusion molecule inhibitor.

23. A method of decreasing growth of a solid tumor in a liver of a subject in need thereof, the method comprising administering to the subject an effective amount of a DNA J-cAMP- dependent PK fusion molecule inhibitor, wherein the inhibitor decreases the size of the solid tumor.

24. The method of claim 22, wherein the liver cancer comprises fibrolamellar hepatocellular carcinoma, hepatoblastoma, hepatocellular carcinoma, hepatocellular adenoma, cavernous hemangioma, or a combination thereof.

25. The method of claim 23, wherein the solid tumor comprises fibrolamellar hepatocellular carcinoma, hepatoblastoma, hepatocellular carcinoma, hepatocellular adenoma, cavernous hemangioma, or a combination thereof.

26. The method of claim 22 or 23, wherein the DNA J-cAMP-dependent PK fusion molecule inhibitor specifically binds to a DNAJB1-PRKACA fusion protein or a fragment thereof.

27. The method of claim 22 or 23, wherein the inhibitor comprises an antibody that specifically binds to a DNAJBl-PRKACA fusion protein or a fragment thereof; a small molecule that specifically binds to a DNAJB 1 protein; a small molecule that specifically binds to a PRKACA protein; an antisense RNA or antisense DNA that decreases expression of a DNAJBl-PRKACA fusion polypeptide; a siRNA that specifically targets a DNAJBl- PRKACA fusion gene; or a combination thereof.

28. The method of claim 27, wherein the small molecule that specifically binds to a PRKACA protein comprises 5-Iodotubercidin, A-3 Hydrochloride, KT 5720, ML-9, H-89, HA- 1004, H7, A-674563, K252, or a combination thereof.

29. A diagnostic kit for determining whether a sample from a subject exhibits a presence of a DNAJBl-PRKACA fusion molecule, the kit comprising at least one oligonucleotide that specifically hybridizes to the DNAJBl-PRKACA fusion molecule, or a portion thereof.

30. The kit of claim 29, wherein the oligonucleotides comprise a set of nucleic acid primers or in situ hybridization probes.

31. The kit of claim 29, wherein the oligonucleotide recognizes a nucleic acid comprising SEQ ID NO: 2.

32. The kit of claim 30, wherein the primers prime a polymerase reaction only when a

DNAJBl-PRKACA fusion is present.

33. The kit of claim 29, wherein the determining comprises gene sequencing, selective

hybridization, selective amplification, gene expression analysis, or a combination thereof.

34. A diagnostic kit for determining whether a sample from a subject exhibits a presence of a DNAJBl-PRKACA fusion protein, the kit comprising an antibody that specifically binds to a DNAJBl-PRKACA fusion protein comprising SEQ ID NOl, wherein the antibody will recognize the protein only when a DNAJBl-PRKACA fusion protein is present.

35. A method for detecting the presence of a DNAJBl-PRKACA fusion in a human subject, the method comprising:

(a) obtaining a biological sample from the human subject; and

(b) detecting whether or not there is a DNAJB 1 -PRKAC A fusion present in the subject.

36. The method of claim 35, wherein the detecting comprises measuring DNAJBl-PRKACA fusion protein levels by ELISA using an antibody directed to SEQ ID NO: 1; western blot using an antibody directed to SEQ ID NO: 1; mass spectroscopy, isoelectric focusing, or a combination thereof.

37. A method for detecting the presence of a DNAJBl-PRKACA fusion in a human subject, the method comprising:

(a) obtaining a biological sample from a human subject; and

(b) detecting whether or not there is a nucleic acid sequence encoding a DNAJB 1 - PRKACA fusion protein in the subject.

38. The method of claim 37, wherein the nucleic acid sequence comprises SEQ ID NO: 2.

39. The method of claim 37, wherein the detecting comprises using hybridization,

amplification, or sequencing techniques to detect a DNAJBl-PRKACA fusion.

40. The method of claim 39, wherein the amplification uses primers directed to SEQ ID NO: 2.