WO2014151734A1 - Fusion proteins and methods thereof - Google Patents

Fusion proteins and methods thereof Download PDF

Info

Publication number
WO2014151734A1
WO2014151734A1 PCT/US2014/026351 US2014026351W WO2014151734A1 WO 2014151734 A1 WO2014151734 A1 WO 2014151734A1 US 2014026351 W US2014026351 W US 2014026351W WO 2014151734 A1 WO2014151734 A1 WO 2014151734A1
Authority
WO
WIPO (PCT)
Prior art keywords
egfr
fusion
fusion protein
protein
gene
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2014/026351
Other languages
English (en)
French (fr)
Inventor
Antonio Iavarone
Anna Lasorella
Raul Rabadan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Columbia University in the City of New York
Original Assignee
Columbia University in the City of New York
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Columbia University in the City of New York filed Critical Columbia University in the City of New York
Priority to CA2907152A priority Critical patent/CA2907152A1/en
Priority to EP14769771.8A priority patent/EP2968551B1/en
Priority to AU2014236947A priority patent/AU2014236947A1/en
Priority to JP2016502111A priority patent/JP2016515508A/ja
Priority to KR1020157029166A priority patent/KR20150129847A/ko
Publication of WO2014151734A1 publication Critical patent/WO2014151734A1/en
Priority to US14/853,568 priority patent/US10208296B2/en
Anticipated expiration legal-status Critical
Priority to US16/246,167 priority patent/US11505788B2/en
Priority to US18/046,131 priority patent/US20230303985A1/en
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03003—Phosphoserine phosphatase (3.1.3.3)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/10—Protein-tyrosine kinases (2.7.10)
    • C12Y207/10001—Receptor protein-tyrosine kinase (2.7.10.1)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators

Definitions

  • Gliobiastoma multiforme is the most common form of brain cancer and among the most incurable and lethal of all human cancers.
  • the current standard of care includes surgery, chemotherapy, and radiation therapy.
  • the prognosis of GBM remains uniformly poor.
  • the target population of GBM patients who may carry EGFR gene fusions and would benefit from targeted inhibition of EGFR kinase activity is estimated to correspond to 6,000 patients per year world-wide.
  • An aspect of the invention is directed to a purified fusion protein comprising a tyrosine kinase domain of an EGFR protein fused to a polypeptide that constitutively activates the tyrosine kinase domain of the EGFR protein.
  • the purified fusion protein is essentially free of other human proteins.
  • An aspect of the invention is directed to a purified fusion protein comprising the tyrosine kinase domain of an EGFR protein fused 5' to a polypeptide comprising a phosphoserine phosphatase (PSPFf) protein.
  • the purified fusion protein is essentially free of other human proteins,
  • An aspect of the invention is directed to a purified fusion protein comprising the tyrosine kinase domain of an EGFR protein fused 3' to a polypeptide comprising a Cullin- associated and neddylation-dissociated (CAND) protein.
  • the CAND protein is CAND 1 , CAND2, or CAND3.
  • the purified fusion protein is essentially free of other human proteins.
  • An aspect of the invention is directed to a purified fusion protein encoded by an EGFR-SEPT 14 nucleic acid, wherein EGFR.-SEPT14 comprises a combination of exons 1-25 of EGFR located on human chromosome 7pl l.2 spliced 5' to a combination of exons 7-10 of SEPT 14 located on human chromosome 7, wherein a genomic breakpoint occurs in any one of exons 1-25 of EGFR and any one of exons 7-10 of SEPTM.
  • the purified fusion protein is essentially free of other human proteins.
  • An aspect of the invention is directed to a purified EGFR-SEPT14 fusion protein comprising SEQ ID NO: 1 or 5.
  • the purified fusion protein is essentially free of other human proteins.
  • An aspect of the invention is directed to a purified EGFR-PSPH fusion protein comprising SEQ ID O: 7 or 1 1.
  • the purified fusion protein is essentially free of other human proteins.
  • An aspect of the in vention is directed to a purified EGFR-PSPH fusion protein having a genomic breakpoint comprising SEQ ID NO: 9.
  • the purified fusion protein is essentially free of other human proteins.
  • An aspect of the invention is directed to a synthetic nucleic acid encoding an EGFR-SEPT 14 fusion protein comprising SEQ ID NO: 2.
  • An aspect of the in vention is directed to a synthetic nucleic acid encoding an EGFR-SEPT! 4 fusion protein having a genomic breakpoint comprising SEQ ID NO: 4.
  • An aspect of the invention Is directed to a synthetic nucleic acid encoding an EGFR-PSPH fusion protein comprising SEQ ID NO: 8.
  • An aspect of the invention is directed to a synthetic nucleic acid encoding an EGFR-PSPH fusion protein having a genomic breakpoint comprising SEQ ID NO: 10,
  • An aspect of the invention is directed to a synthetic nucleic acid encoding an EGFR-CAND 1 fusion protein comprising SEQ ID NO: 14.
  • An aspect of the in vention is directed to an antibody or antigen-binding fragment thereof that specifically binds to a purified fusion protein comprising a tyrosine kinase domain of an EG FR protein fused to a polypeptide that constiiutively activates the tyrosine kinase domain of the EGFR protein.
  • the fusion protein is an EGFR- SEPT fusion protein, an EGFR-PSPH fusion protein, or an EGFR-CAND fusion protein
  • the EGFR-SEPT fusion protein is EGFR-SEPT 14.
  • the EGFR-SEPT fusion protein comprises the amino acid sequence of SEQ ID NO: I, 3, or 5.
  • the EGFR-CAND fusion protein is EGFR-CAND 1.
  • the EGFR-CAND fusion protein comprises the amino acid sequence of SEQ ID NO: 13, 15, or 17
  • An aspect of the in vention is directed to an antibody or antigen-binding fragment thereof that specifically binds to a purified fusion protein comprising a tyrosine kinase domain of an EGFR protein fused to a polypeptide comprising the coiied-coil domain of a Septin protein.
  • the EGFR-SEPT tusion protein is EGFR-SEPT 14.
  • the EGFR-SEPT fusion protein comprises the amino acid sequence of SEQ ID NO: 1, 3, or 5.
  • An aspect of the invention is directed to an antibody or antigen-binding fragment thereof, that specifically binds to a purified tusion protein comprising a tyrosine kinase domain of an EGFR protein fused to a polypeptide comprising a Cuilin-associated and neddylation-dissociated (CAND) protein.
  • the EGFR-CAND fusion protein is EGFR-CAND 1.
  • the EGFR-CAND iusion protein comprises the amino acid sequence of SEQ ID NO: 13, 15, or 17
  • the inhibitor comprises an antibody that specifically binds to an EGFR-SEPT fusion protein, an EGFR-PSPH fusion protein, an EGFR-CAND fusion protein, or a fragment thereof; a small molecule that specifically binds to an EGFR protein: an antisense K A or antisense DNA that decreases expression of an EGFR-SEPT fusion protein, an EGFR-PSPH fusion protein, an EGFR-CAND fusion; a siRNA that specifically targets an EGFR-SEPT fusion gene, an EGFR-PSPH fusion gene, or an EGFR-CAND; or a combination thereof.
  • the CAND protein is CAND1.
  • the SEPT protein is SEPT 14.
  • the small molecule thai specifically binds to an EGFR protein comprises AZD4547, NVP-BGJ398, PD173074, NF449, TK1258, BIBF-1120, BMS- 582664, AZD-2171 , TSU68, AB1010, AP24534, E-7080, LY2874455, or a combination thereof.
  • An aspect of the invention is directed to a method for treating a gene- fusion associated cancer in a subject in need thereof, the method comprising administering to the subject an effective amount of an EGFR fusion molecule inhibitor, in one embodiment, the gene-fusion associated cancer comprises glioblastoma multiforme, breast cancer, lung cancer, prostate cancer, or colorectal carcinoma.
  • the EGFR fusion comprises an EGFR protein fused to a polypeptide that constitutively activates the tyrosine kinase domain of the EGFR protein.
  • the EGFR fusion protein is an EGFR-SEPT 14 fusion protein, an EGFR-PSPH fusion protein, or an EGFR-CANDl fusion protein.
  • the inhibitor comprises an antibody that specifically binds to an EGFR-SEPT fusion protein, an EGFR-PSPH fusion protein, an EGFR-CAND fusion protein, or a fragment thereof: a small molecule that specifically binds to an EGFR protein; an antisense RNA or antisense DNA that decreases expression of an EGFR-SEPT fusion protein, an EGFR-PSPH fusion protein, an EGFR-CAND fusion; a siRNA that specifically targets an EGFR-SEPT fusion gene, an EGFR-PSPH fusion gene, or an EGFR-CAND; or a combination thereof.
  • the small molecule that specifically binds to an EGFR protein comprises AZD4547, NVP-BGJ398, PD ! 73074, NF449, TK1258, BIBF-1120, BMS-582664, AZD- 2171, TSU68, ABlOiO, AP24534, E-7080, LY2874455, or a combination thereof.
  • An aspect of the invention is directed to a method of decreasing growth of a solid tumor in a subject in need thereof, the method comprising administering to the subject an effective amount of an EGFR fusion molecule inhibitor, wherein the inhibitor decreases the size of the solid tumor.
  • the subject is afflicted with a gene-fusion associated cancer.
  • the gene-fusion associated cancer comprises glioblastoma multiforme, breast cancer, lung cancer, prostate cancer, or colorectal carcinoma.
  • the solid tumor comprises glioblastoma multiforme, breast cancer, lung cancer, prostate cancer, or colorectal carcinoma.
  • the EGFR fusion comprises an EGFR protein fused to a.
  • the EGFR fusion protein is an EGFR-SEPT! 4 fusion protein, an EGFR-PSPH fusion protein, or an EGFR-CANDl fusion protein.
  • the inhibitor comprises an antibody that specifically binds to an EGFR-SEPT fusion protein, an EGFR-PSPH fusion protein, an EGFR-CAND fusion protein, or a fragment thereof; a small molecule that specifically binds to an EGFR protein; an antisense RNA or antisense DNA that decreases expression of an EGFR-SEPT fusion protein, an EGFR-PSPH fusion protein, an EGFR-CAND fusion; a siRNA that specifically targets an EGFR-SEPT fusion gene, an EGFR-PSPH fusion gene, or an EGFR-CAND; or a combination thereof.
  • the small molecule that specifically binds to an EGFR protein comprises AZD4547, NVP-BGJ398, PD173074, NF449, TK1258, BIBF-1 120, BMS-582664, AZD-2171, TSU68, AB1010, AP24534, E-7080, LY2874455, or a combination thereof.
  • the EGFR fusion protein is an EGFR-SEPT 14 fusion protein, an EGFR- PSPH fusion protein, or an EGFR-CANDI fusion protein.
  • the inhibitor comprises an antibody that specifically binds to an EGFR-SEPT fusion protein, an EGFR- PSPH fusion protein, an EGFR-CAND fusion protein, or a fragment thereof; a small molecule that specifically binds to an EGFR protein; an antisense R A or antisense DNA that decreases expression of an EGFR-SEPT fusion protein, an EGFR-PSPH fusion protein, an EGFR-CAND fusion; a siRNA that specifically targets an EGFR-SEPT fusion gene, an EGFR-PSPH fusion gene, or an EGFR-CAND; or a combination thereof.
  • the small molecule that specifically binds to an EGFR protein comprises AZD4547, NVP-BGJ398, PD 173074, NF449, TK1258, BIBF-1120, BMS-582664, AZD- 2171, TSU68, AB1010, AP24534, E-7080, LY2874455, or a combination thereof.
  • the fusion protein is an EGFR-SEPT 14 fusion protein, an EGFR-PSPH fusion protein, or an EGFR-CANDI fusion protein.
  • the determining comprises gene sequencing, selective hybridization, selective amplification, gene expression analysis, or a combination thereof.
  • An aspect of the invention is directed to a diagnostic kit for determining whether a sample from a subject exhibits a presence of an EGFR fusion protein, the kit comprising an antibody that specifically binds to an EGFR fusion protein comprising SEQ ID NO: 1, 3 , 5, 7, 9, 1 1 , 13, 15, or 17, wherein the antibody will recognize the protein only when an EGFR fusion protein is present.
  • the fusion protein is an EGFR-SEPT14 fusion protein, an EGFR-PSPH fusion protein, or an EGFR-CAND1 fusion protein.
  • the subject is afflicted with a gene-fusion associated cancer. In one
  • the gene-fusion associated cancer comprises glioblastoma multiforme, breast cancer, lung cancer, prostate cancer, or colorectal carcinoma.
  • An aspect of the invention is directed to a method for detecting the presence of an EGFR fusion in a human subject.
  • the method comprises obtaining a. biological sample from the human subject: and detecting whether or not there is an EGFR fusion present in the subject.
  • the detecting comprises measuring EGFR fusion protein levels by ELISA using an antibody directed to SEQ ID MO: 1, 3, 5, 7, 9, 1 1, 13, 15, or 17: western blot using an antibody directed to SEQ ID MO: 1 , 3, 5, 7, 9, 11 , 13, 15, or 17: mass spectroscopy, isoelectric focusing, or a combination thereof.
  • An aspect of the invention is directed to a method for detecting the presence of an EGFR fusion in a human subject.
  • the method comprises obtaining a biological sample from a human subject; and detecting whether or not there is a nucleic acid sequence encoding an EGFR fusion protein in the subject.
  • the nucleic acid sequence comprises any one of SEQ ID MOS: 2, 4, 8, 10, 14, and 16.
  • the detecting comprises using hybridization, amplification, or sequencing techniques to detect an EGFR fusion.
  • the amplification uses primers comprising SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29.
  • the fusion protein is an EGFR-SEPT14 fusion protein, an EGFR-PSPH fusion protein, or an EGFR-CAND1 fusion protein.
  • FIG. 1A is a chromosome view of validated GBM genes scoring at the top of each of the three categories by MutComFocal. The plot shows mutated genes without significant copy number alterations (Mut, mutation %, frequency of mutations). Previously known GBM genes are indicated in green (light grey in black and white image), new and
  • GBM genes are indicated in red (dark grey in black and white image).
  • FIG, IB is a chromosome view of validated GBM genes scoring at the top of each of the three categories by MutComFocal.
  • the plot shows mutated genes in regions of focal and recurrent amplifications (Amp-Mut, Amplification/mutation scores).
  • Previously known GBM genes are indicated in green (light grey in black and white image), new and
  • GBM genes are indicated in red (dark grey in black and white image).
  • FIG. 1C is a chromosome view of validated GBM genes scoring at the top of each of the three categories by MutComFocal.
  • the plot shows mutated genes in regions of focal and recurrent deletions (Del-Mut, Deletion/mutation scores).
  • Previously known GBM genes are indicated in green (light grey in black and white image), new and independently validated GBM genes are indicated in red (dark grey in black and white image).
  • FIG. 2A-B shows Localization of altered residues in LZTR-1.
  • FIG. 2A shows lysates from 293T ceils transfeeted with vectors expressing LZTR-1 and the Flag-Cul3 wild type (VVT), Flag-Cul3 -dominant negative (DN) or the empty vector were immimoprecipitated with Flag antibody and assayed by western blot with the indicated antibodies. *, non specific band; left bracket indicates Cul3 polypeptides. The molecular weight is indicated on the right.
  • FIG. 2B shows homology model of the Kelch (green; grey in black and white image of left hand side of ribbon diagram), BTB (cyan; (light grey in black and white image of center and right of ribbon diagram) and BACK, (purple; dark grey in black and white image of center and right of ribbon diagram) domains of LZTR-1 with the Cul3 N-terminal domain (white) docked onto the putative binding site.
  • GBM mutations are indicated in red (dark grey in black and white image; left hand side of ribbon diagram).
  • FIG, 2C Sequence alignment of the six blades from the Kelch ⁇ -propeller domain. Each blade contains four core ⁇ -strands, labeled a, b, e, d. conserveed residues are highlighted in gray and residues mutated in GBM are shown in red. Insertions at the end of blades 5 and 6 are indicated in brackets.
  • FIGS. 3C-I Loss of C» ® »D» drives mesenchymal transformation of GBM.
  • 3c Kaplan-Meier analysis for glioma patients with low CTNND2 mRNA expression ( ⁇ 2-fold, red line) compared with the rest of glioma (blue line).
  • 3d Kaplan-Meier analysis for glioma patients with low CTNND2 mRNA expression ( ⁇ 2-fold) and decreased CTNND2 gene copy number ( ⁇ 1) (red line) compared with the rest of glioma (blue line).
  • FIG. 4A EGFR-SEPT14 gene fusion identified by whole transeriptome sequencing. Split reads are shown aligning on the breakpoint. The predicted reading frame at the breakpoint is shown at the top with EGFR sequences in blue and SEPT 14 in red.
  • the amino acid sequence (TOP) is SEQ ID MO: 1 ; the nucleotide sequence (bottom) is SEQ ID NO: 2.
  • FIG. 4B EGFR-SEPT14 gene fusion identified by whole transcriptome sequencing, (left panel), E GFRSEPT14- specific PGR from cDNA derived from GBMs. Marker, 13d ladder, (right panel), Sanger sequencing chromatogram showing the reading frame at the breakpoint (SEQ ID NO: 4) and putative translation of the fusion protein (SEQ ID NO: 3) in the positive sample.
  • FIG. 4C EGFR-SEPT14 gene fusion identified by whole transcriptome sequencing.
  • EGFR-Septinl4 fusion protein sequence (SEQ ID NO: 5) and schematics. Regions corresponding to EGFR and Septin14 are shown in blue (left hand side of diagram; (grey in black and white image; sequence comprising "MRP...VIQ” amino acids of SEQ ID NO: 5) and red (right hand side of diagram; light grey in black and white image; sequence comprising "LQD...RKK” amino acids of SEQ ID NO: 5), respectively.
  • the fusion joins the tyrosine kinase domain of EGFR and the Coiled-coil domain of Septin14.
  • FIG, 4D EGFR-SEPT14 gene fusion identified by whole transcriptome sequencing. Genomic fusion of EGFR exon 25 with intron 9 oi SEPT 14. In the fuse niR A exon 24 of EGFR is spliced 5' to exon 10 of SEPT14. Solid arrows indicate the position of the fusion genome primers that generate a fusion specific PGR product in the GBM sample TCGA-27-1837.
  • FIG, 5A Expression of EGFR-SEPTl 4 fusion promotes an aggressive phenotype and inhibition of EGFR kinase delays GBM growth in vivo. Growth rate of SNB19 glioma cells transduced with a lentivirus expressing EGFR-SEPTl.4, EGFR Viii, EGFR WT or the empty vector (average of triplicate cultures).
  • FIG. SB Expression of EGFR-SEPTl 4 fusion promotes an aggressive phenotype and inhibition of EGFR kinase delays GBM growth in vivo.
  • FIG. 5C Expression of EGFR-SEPTl 4 fusion promotes an a ggressive phenotype and inhibition of EGFR kinase delays GBM growth in vivo. Quantification of the cell co vered area for the experiments shown in b (a verage of triplicate cultures). All error bars are SD.
  • FIG. 6 shows the distribution of substitutions from whole exome data.
  • the two BTB-BAC domains of LZTR-1 are included along with the predicted secondary structure from HHpred°.
  • the 3 -box is the Cul3 binding element within the BACK domain.
  • the secondary structure of KLHL3 (PDB ID 4HXI), KLHL1 1 (PDB ID 4AP2) and Gigaxonin (PDB ID 3HVE) are based on the crystal structures.
  • the secondary structure of SPOP is based on a crystal structure for the BTB and 3 -box region (PDB ID 3 HTM) and HHpred predictions from the remainder of the BACK domain. Only the N -terminal half of the BACK domain from KLHL3, KLHL1 1 and Gigaxonin is included, as SPOP and LZTR-1 contain truncated versions of the BACK domain.
  • FIG. iOB Pattern of somatic mutations, C Vs and expression of CTNND2 in GBM. Somatic deletions of CTNND2. Samples are sorted according to the focality of CTNND2 deletion. In the red-blue scale, white corresponds to normal (diploid) copy number, blue is deletion and red is gain.
  • FIG, 101 Pattern of somatic mutations, CNVs and expression of CTNND2 in GBM.
  • CTNND2 mRNA expression analysis from Atlas-TCGA samples shows that CTNND2 is significantly down -regulated in the mesenchymal subgroup. In the green-red scale, black is the median, green is down-regulation and red is up-regulation.
  • FIG. 11 A EGFR-PSPH gene fusion identified by whole transcriptome sequencing. Split reads are shown aligning on the breakpoint. The predicted reading frame at the breakpoint is shown at the top with EGFR sequences in blue (grey in black and white image; encompassing "SRR..VIQ” amino acids and “AGT...CAG” nucleotides) and PSPH in red (light grey in black and white image; encompassing "DAF . . .QQV” amino acids and "GAT, , .CAA” nucleotides).
  • the amino acid sequence (TOP) is SEQ ID NO: 7; the nucleotide sequence (bottom) is SEQ ID NO: 8.
  • FIG, 11B EGFR-PSPH gene fusion identified by whole transcriptome sequencing, (left panel), EGFR-PSHP specific PGR from cDN A derived from GBMs, Marker, Ikb ladder, (right panel), Sanger sequencing chromatogram showing the reading frame (SEQ ID NO: 10) at the breakpoint and putative translation of the fusion protein (SEWQ ID NO: 9) in the positive sample.
  • FIG, I IC EGFR-PSPH gene fusion identified by whole transcriptome sequencing.
  • EGFR-PSPH fusion protein sequence SEQ ID NO: 1 1
  • schematics Regions corresponding to EGFR and PSPH are shown in blue (grey in black and white image; left hand side of schematic; sequence comprising "MRP...VIQ” amino acids of SEQ ID NO: 1 1) and red (light grey in black and white image: right hand side of schematic, encompassing amino acids "DAF...LEE” of SEQ ID NO: 11.), respectively.
  • the fusion includes the tyrosine kinase domain of EGFR and the last 35 amino acids of PSPH.
  • FIG. I2A NFASC-NTRK1 gene fusion identified by whole transcripiome sequencing. Split reads are shown aligning on the breakpoint. The predicted reading frame at the breakpoint is shown at the top with NFASC sequences in bine (grey in black and white image; encompassing "RVQ...GED” amino acids and “AGA...ATT” ucleotides) and NTR l in red (light grey in black and white image; encompassing ' ⁇ ...VGL” amino acids and "AGA...AAG” nucleotides).
  • FIG. 12B NFASC-NTRK1 gene fusion identified by whole transcriptome sequencing, (left panel), NFA SC-NTRK1 specific PGR from cDNA derived from GBMs. Marker, ! kb ladder, (right panel), Sanger sequencing chromatogram showing the reading frame at the breakpomt and putative translation of the fusion protein in the positive sample.
  • FIG, 12C NFASC-NTRK1 gene fusion identified by whole transcriptome sequencing. NFASC-NTR 1 fusion protein sequence and schematics. Regions
  • the fusion includes two of the five fibroneeiin-type III domain of neurofascin and the protein kinase domain of NTRKl .
  • FIG, 12D NFA SC -NTRKl gene fusion identified by whole transcriptome sequencing. Genomic fusion of NFASC intron 9 with intron 21 of NTRKL In the fuse mRNA exon 21 of NFASC is spliced 5' to exon 10 of NTRKl. Solid arrows indicate the position of the fusion genome primers that generate a fusion specific PGR product in the GBM sample TCGA-06-541 1.
  • FIG. 13 shows the expression measured by read depth from RNA-seq data. Note the very high level of expression in the regions of the genes implicated in the fusion events.
  • FIG, 14A CAND l -EGFR gene fusion identified by whole transcriptome sequencing. Split reads are shown aligning on the breakpoint. The predicted reading frame at the breakpoint is hown at the top with CANDl sequences in blue (grey in black and white image; sequence comprising "TSA...LSR" amino acids of SEQ ID NO: 13 and
  • TTA...CAG nucleotides of SEQ ID NO: 14
  • EGFR in red (light grey in black and white image; sequence comprising "CTG...VGX” amino acids of SEQ ID NO: 13 and "ATC...GGC” nucleotides of SEQ ID NO: 14).
  • the amino acid sequence (TOP) is SEQ ID NO: 13; the nucleotide sequence (bottom) is SEQ ID NO: 14.
  • FIG. 14B CANDl -EGFR gene fusion identified by whole iranscriptome sequencing, (left panel), CANDl-EGFR specific PGR from cDNA der ved from GBMs. Marker, lkb ladder, (right panel), Sanger sequencing c romatogram showing the reading frame at the breakpoint (SEQ ID NO: 15) and putative translation of the fusion protein (SEQ ID NO: 16) in the positive sample.
  • FIG. 14C CANDl-EGFR gene fusion identified by whole transcriptome sequencing.
  • CANDl-EGFR fusion protein sequence SEQ ID NO: 12). Regions
  • CAND1 and EGFR are shown in blue (grey in black and white image; sequence comprising "MAS...LSR” amino acids of SEQ ID NO: 12) and red (grey in black and white image; sequence comprising "CTG...IGA*” amino acids of SEQ ID NO: 12), respectively.
  • FIG, 14D CAND l -EGFR gene fusion identified by whole transcriptome sequencing. Genomic fusion of CAND1 intron 4 with intron 15 of EGFR. In the fuse mRNA exon 4 of C AND 1 is spliced 5' to exon 16 of EGFR,
  • FIG. 15 is a photographic image of a blot showing the interaction with Cu33 and protein stability of wild type and mutant LZTR-1. Lysates from SF188 glioma cells transfected with vectors expressing Myc-LZTR-l. and Flag-Cul3 or the empty vector were immunoprecipitated with Flag antibody and assayed by western blot with the indicated antibodies. *, non specific band; arrowhead indicates neddylated CuI3.
  • FIG, I6B is a photographic image of a blot showing the interaction with Cui3 and protein stability of wild type and mutant LZTR-1. Stea dy state protein levels of wild type LZTR-1 and GB -related mutants.
  • FIG. 1 D is a photographic image of a blot showing the interaction with Cul3 and protein stability of wild type and mutant LZTR-I , Semi-qua titative RT-PCR evaluation of LZTR-1 wild type and LZTR-1 -R810W ' SNA expression in cells transfected as in FIG. 16C.
  • FIG, 17A is a graph showing functional analysis of LZTR- 1 wild type and GBM associated mutants in GBM-derived cells.
  • FIG. 17C is a linear regression plot of in vitro limiting dilution assay using GBM- derived glioma spheres #46 expressing vector or LZTR-1.
  • the frequency of sphere forming cells was 8.49 ⁇ 1.04 and 1.44 ⁇ 0.05% in vector and LZTR- 1 expressing cells, respectively (p 0.00795).
  • Each data point represents the average of triplicates. Error bars are SD.
  • FIG. 17D is a graph and photographic microscopy images showing functional analysis of LZTR-1 wild type and GBM associated mutants in GBM-derived cells.
  • Left upper panels Bright field microphotographs of GBM-derived line 46 cells six days after transduction with vector or LZTR-1 expressing lenti virus.
  • Left lower panels Bright field microphotographs of spheres from GBM-derived glioma cells #46 expressing lentivirus expressing vector or LZTR-1 from experiment in FIG. 17C.
  • FIG. 17E is a photographic image of a western blot analysis of GBM-derived cells #84 expressing vector or LZTR-1.
  • FIG. 17F is a linear regression plot of in vitro limiting dilution assay using GBM- derived line 84 expressing vector, LZTR-1 , LZTR- 1-R810W or LZTR-1 -W437STOP.
  • the frequency of sphere forming cells was 7.2 ⁇ 0.92 for vector, 1.48 ⁇ 0.09 for LZTR-1 wild type ⁇ /> - 0.0096): 7.82+0.99 for LZTR-1 -R810W (p - 0.2489); and 6.74 : I 07 for LZTR-1 -- W437STOP (p ------ 0.2269). Error bars are SD.
  • FIGS. 18A-B are photographic microscopy images showing expression of 8- catenin in neurons and ⁇ -catenin driven loss of mesenchymal marker in GBM.
  • FIG. ISA shows a. pattern of expression of ⁇ -catenin in the developing brain, as determined by iramunostaining. Double inimiinoiluorescence staining of brain cortex using ⁇ -caienin antibody (red; dark grey in black and white image (center)) and ⁇ -tubulin (green; light grey- in black and white image (right)); Nuclei are counterstained with Dapi (blue; grey in black and white image (Left)).
  • FIG. ISA shows a. pattern of expression of ⁇ -catenin in the developing brain, as determined by iramunostaining. Double inimiinoiluorescence staining of brain cortex using ⁇ -caienin antibody (red; dark grey in black and white image (center)) and ⁇ -tubulin (green; light grey
  • FIG. 18C is a photographic image of a western blot using the indicated antibodies for U87 cells expressing ⁇ -catenin wild type, glioma-associated ⁇ -catenin mutants or the empty vector.
  • FBN fibronectin. Vinculin is shown as control for loading.
  • FIGS. 19A-B show a functional analysis of ⁇ -catenin in mesenchymal GBM.
  • FIG. 19 A is a photographic microscopy image of immunofluorescence for fibronectin.
  • FIG. 19C is a bar graph showing the quantification of fluorescence intensity for ⁇ -tubulin in cells #48 infected with lentiviruses expressing CTNND2 or the empty vector.
  • FIG. I9D are photographic microscopy images showing time course analysis of ⁇ -tubulin expression in glioma spheres #48 transduced with lentiviruses expressing CTNND2 or the empty vector. Note the loss from the advanced culture of ⁇ -Tubuliii expressing cells.
  • FIGS. 19E-F are graphs.
  • FIG. 19F shows a longitudinal analysis of bioluminescence imaging in mice injected intracranially with GBM-derived line 48 expressing vector or ⁇ -caienia n - 3 mice for vector and 5 for ⁇ -catenin. Data are mean ⁇ SEM of pho ton counts.
  • FIGS. 20A-E show the functional analysis of EGFR-SEPT 14 fusion and effect of inhibition of EGFR kinase on glioma growth.
  • FIG. 20B is a western blot analysis of GBM-derived primary cells (#48) expressing vector, EGFR Viii or EGFR-SEP14 fusion cultured in the presence of EGF.
  • FIG. 20C is a photohraphic image of a blot showing GBM-derived cells (#48) expressing vector, EGFR Viii or EGFR-SEP14 fusion that were cultured in the absence of EGF for 48 h and then stimulated with EGF 2()ng/ml for the indicated time. Cells were assayed by western blot using the indicated antibodies.
  • FIG. 20D is a graph of GSEA showing up-regulation of STAT3 target genes in primary human GBM carrying the EGFR-SEPT 14 fusion gene
  • FIG. 20E is a bar graph showing the survival of GBM- derived cells (#48) expressing vector, EGFR wild type, EGFR Viii or EGFR-SEP14 fusion after treatment with lapatinib for 48 h at the indicated concentrations. Data are MearcfcSD of triplicate samples.
  • FIG. 21 is a plot showing the number of mutations in TCGA samples harboring MutComFocal gene candidates.
  • the number of mutations M8 was plotted in samples harboring G as solid circles.
  • the mean of M8 is also plotted as asterisks.
  • the 95% confidence interval of a sample being hyper-mutated (1 1 ⁇ 1.96*a) was plotted and shown that for all G, the mean of M8 falls well within the 95% confidence interval, demonstrating that MutComFocal genes do not tend to occur in hypermutated samples,
  • FIGS. 22A-B show pattern of somatic mutations, CNVs and expression of CTNND2 in GBM.
  • FIG. 22A are photograpgic microscopy images of immunofluorescence staining of human primary GBM included in tiss ue microarrays (TMA) using ⁇ -catenin antibody (red; darkt grey in black and white image); Nuclei are counterstained with Dapi (blue; grey in black and white image). Two representative ⁇ -catenin-positive and two ⁇ - catenin-negative tumors are shown in the upper and lower panels, respectively.
  • FIG. 22B is a Western Blot analysis of the expression of ⁇ -catenin in a panel of GBM-derived glioma sphere cultures. Brain, normal human brain. Arrowhead indicated ⁇ -eatenin; Asterisk, nonspecific band. Vinculin is shown as control for loading.
  • overexpression or overactivity have been associated with a number of cancers, including lung cancer, anal cancers and glioblastoma multiforme.
  • Phosphoserine phosphatase is an enzyme responsible for the third and last step in L-serine formation. It catalyzes magnesium-dependent hydrolysis of L-phosphoserine and is also involved in an exchange reaction between L-serine and L-phosphoserine.
  • a protein is encoded by a nucleic acid (including, for example, genomic DMA, 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.
  • a fusion protein of the invention comprises a tyrosine kinase domain of an EGFR protein fused to a polypeptide that constitutive! ⁇ ' activates the tyrosine kinase domain of the EGFR protein.
  • the fusion protein can be an EGFR-SEPT fusion protein, an EGFR-PSPH fusion protein, or an EGFR-CAND fusion protein.
  • Genbank ID for the EGFR gene is 1956.
  • Four isoforms are listed for EGFR, e.g., having Genebank Accession Nos, NP_ 005219 (corresponding nucleotide sequence NM_005228); NP . 958439 (corresponding nucleotide sequence NM 201282); NP_ 958440 (corresponding nucleotide sequence MJ201283); NP_958441 (corresponding nucleotide sequence M_201284).
  • the nucleotide and amino acid sequences can be readily obtained by one of ordinary skill in the art using the listed accession numbers.
  • Genbank ID for the PSPH gene is 5723.
  • the Genebank Accession No. for PSPH is NP 004568 (corresponding nucleotide sequence NM 004577).
  • the nucleotide and amino acid sequences can be readily obtained by one of ordinary skill in the art using the listed accession numbers.
  • an "EGFR fission molecule” can be a nucleic acid which encodes a polypeptide corresponding to a. fusion protein comprising a tyrosine kinase domain of an EGFR protein fused to a polypeptide that constitutively activates the tyrosine kinase domain of the EGFR protein.
  • an EGFR fusion molecule can include an EGFR-containing fusion comprising the amino acid sequence corresponding to Genebank Accession no. NP__ 005219, NP_958439, NP_958440, or NP__958441.
  • AN EGFR fusion molecule can also include a tyrosine kinase domain of an EGFR protein fused to a protein encoded by any one of the genes lis ted in Table 10.
  • AN EG FR fusion molecule can include a variant of the above described examples, such as a fragment thereof.
  • 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 DMA 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 and include, but are not limited to, histones and methylation.
  • a. nucleic acid encoding anan EGFR EGFR 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.
  • the invention further provides for nucleic acids that are complementary to an EGFR fusion molecule.
  • Complementary nucleic acids can hybridize to the nucleic acid sequence described above under stringent hybridization conditions.
  • stringent hybridization conditions include temperatures above 30°C, above 35°C, in excess of 2°C, and/or salinity of less than about 500 mM, or less than 200 niM.
  • 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.
  • protein variants can include amino acid sequence modifications.
  • amino acid sequence modifications fall into one or more of three classes: substitutional, insertionai or deietionai 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.
  • an EGFR fusion molecule comprises a protein or polypeptide encoded by a nucleic acid sequence encoding an EGFR fusion molecule, suc as the sequences shown in SEQ ID NOS: 2, 4, 8, 10, 14, or 16.
  • the nuceleic acid sequence encoding an EGFR 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 NOS: 2, 4, 8, 10, 14, or 16.
  • 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 an EGFR fusion molecule is the polypeptide having the amino acid sequence shown in SEQ ID NOS: 1, 3, 5, 7, 9, 1 1 , 13, 15, or 17.
  • the EGFR 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 NOS: 1, 3, 5, 7, 9, 1 1, 13, 15, or 17.
  • an EGFR fusion molecule can be a fragment of an EGFR fusion protein.
  • the EGFR fusion molecule can encompass any portion of at least about 8 consecutive amino acids of SEQ TD NOS: 1 , 3, 5, 7, 9, 11, 13, 15, or 17.
  • 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, at least about 50 amino acids, at least about 60 amino acids, or at least about 75 amino acids of SEQ ID NOS: 1 , 3, 5, 7, 9, 1 1 , 13, 15, or 17.
  • Fragments include all possible amino acid lengths between about 8 and about 100 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.
  • Nucleic acid sequences encoding an EGFR fusion molecule can be synthesized, in whole or in part, using chemical methods known in the art.
  • an EGFR-PSPH fusion protein is a polypeptide having the amino acid sequence shown in SEQ ID NO: 7, 9, or 1 1.
  • An example of an EGFR-CAND fusion protein is EGFR-CAND 1, In one embodiment, an EGFR-CAND 1 fusion polypeptide can have the amino acid sequence shown in SEQ ID NO: 13, 15, or 17.
  • Q J_ajnj ⁇ A polypeptide encoded by a nucleic acid, such as a nucleic acid encoding an EGFR 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.
  • a nucleic acid fragment of an EGFR fusion molecule can encompass any portion of at least about 8 consecutive nucleotides of SEQ ID NOS: 2, 8, or 14.
  • 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, 8, or 14.
  • 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.
  • Methods for producing labeled hybridization or PGR probes for detecting sequences related to nucleic acid sequences encoding a protein, such as EGFR fusion molecule include, but are not limited to, oiigolabeiing, nick translation, end-labeling, or PGR amplification using a labeled nucleotide.
  • nucleic acid sequences such as nucleic acids encoding an EGFR fusion molecule, can be cloned into a vector for the production of an mRNA probe. Such vectors are known in the art.
  • a fragment can be a fragment of a protein, such as an EGFR fusion protein.
  • a fragment of an EGFR fusion can encompass any portion of at least about 8 consecutive amino acids of SEQ TD NOS: 1 , 3, 5, 7, 9, 1 1, 13, 1 5, or 17.
  • 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 an EGFR fusion molecule.
  • Mammalian cells can contain an expression vector (for example, one that contains a nucleic acid encoding a fusion protein comprising a tyrosine kinase domain of an EGFR protein fused to a polypeptide that constitutively activates the tyrosine kinase domain of the EGFR protein) via introducing the expression vector into an appropriate host cell via methods known in the art.
  • An exogenous nucleic acid can be introduced into a cell via a variety of techniques known in the art, such as iipofection, microinjection, calcium phosphate or calcium chloride precipitation, DEAE-dextran-niediaied transfeetion, 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.
  • 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.
  • primary and secondary cells include epithelial ceils, neural ceils, endothelial cells, glial cells, fibroblasts, muscle ceils (such as myoblasts) keratinocytes, formed elements of the blood (e.g., lymphocytes, bone marrow cells), and precursors of these somatic cell types.
  • 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 ceil type of interest.
  • a punch biopsy or removal e.g., by aspiration
  • a source of cancer ceils for example, glioma cells, neuroblastoma cells, and the like.
  • a mixture of primary cells can be obtained from the tissue, using m ethods readily practiced in the art, such as explaining or enzymatic digestion (for examples using enzymes such as pronase, trypsin, collagenase, elastase dispase, and chymoirypsin).
  • 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.
  • 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 an isolated or 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 ceil types plated for the first time; and cell culture suspensions derived from these plated cells.
  • tissue culture substrate for example, flask or dish
  • Secondary cells can be plated primary cells that are removed from the culture substrate and replaled, 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 an EGFR fusion molecule. Ceil Culturing
  • 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 ceil selected. Commercially available medium can be utilized. Non-limiting examples of medium include, for example, Minimal Essential Medium (MEM, Sigma, St.
  • CD-CHO Medium (Invitrogen, Carlsbad, Calif.).
  • 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.
  • Ceil 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.
  • 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 ampicillm; (7) cell protective agents, for example pluronic poiyol; and (8) galactose.
  • soluble factors can be added to the culturing medium.
  • the mammalian cell culture that can be used with the present invention is prepared in a. medium suitable for the ty e of cell being cultured.
  • 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)).
  • the medium can be a conditioned medium to sustain the growth of host cells.
  • cationic polymers include but are not limited to, chitosan or polylysine, (Peppas et al., (2QQ6) Adv Mater. 18: 1345-60; Hoffman, A. S., (2002) Adv Drug Deliv Rev. 43: 3-12;
  • amphipathic polymers can include, but are not limited to collagen, gelatin, fibrin, and carboxymethyl chitin.
  • neutral polymers can include dextran, agarose, or uilulan, (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).
  • Ceils to be cultured can harbor introduced expression vectors, such as plasmids.
  • the expression vector constructs can be introduced via transformation, microinjection, transfeciion, lipofection, electroporatxon, 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.
  • the invention provides methods for use of compounds that decrease the expression level or activity of an EFGR EGFR fusion molecule in a subject.
  • the invention provides methods for using compounds for the treatment of a gene-fusion associated cancer.
  • the gene-fusion associated cancer comprises glioblastoma multiforme, breast cancer, lung cancer, prostate cancer, or colorectal carcinoma.
  • an "EGFR fusion molecule inhibitor” refers to a compound that interacts with an EGFR fusion molecule of the invention and modulates its activity and/or its expression. For example, the compound can decrease the activity or expression of an EGFR fusion molecule.
  • the compound can be an antagonist of an EGFR fusion molecule (e.g., an EGFR fusion molecule inhibitor).
  • Some non-limiting examples of EGFR fusion molecule inhibitors include peptides (such as peptide fragments comprising an EGFR fusion molecule, or antibodies or fragments thereof), small molecules, and nucleic acids (such as siRNA or antisense RNA specific for a nucleic acid comprising an EGFR fusion molecule).
  • Antagonists of an EGFR fusion molecule decrease the amount or the duration of the activity of an EGFR fusion protein.
  • the fusion protein comprises a tyrosine kinase domain of an EGFR protein fused to a polypeptide that constitutively activates the tyrosine kinase domain of the EGFR protein (e.g., EGFR-SEPT (such as EFGR-SEPT14), EGFR-PSPH, or EGFR-CAND (such as EGFR-CAND1)).
  • Antagonists include proteins, nucleic acids, antibodies, small molecules, or any other molecule which decrease the activity of an EGFR fusion molecule.
  • modulate,' ' ' refers to a change in the activity or expression of an EGFR fusion molecule.
  • modulation can cause a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of an EGFR fusion molecule, such as an EGFR fusion protein.
  • an EGFR fusion molecule inhibitor can be a peptide fragment of an EGFR fusion protein that binds to the protein itself.
  • the EGFR fusion polypeptide can encompass any portion of at least about 8 consecutive amino acids of SEQ ID NOS: 1 , 3, 5, 7, 9, 1 1, 13, 15, or 17.
  • 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 200 consecutive amino acids, at least about 300 consecutive amino acids, at least about 400 consecutive amino acids, at least about 500 consecutive amino acids, at least about 600 consecutive amino acids, at least about 700 consecutive amino acids, or at least about 800 consecutive amino acids of SEQ ID NOS: 1, 3, 5, 7, 9, 1 1 , 13, 15, or 17.
  • 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 EGFR fusion peptide fragments can be isolated from a natural source, genetically engineered, or chemically prepared. These methods are well .known in the art.
  • An EGFR fusion molecule inhibitor can be a protein, such as an antibody
  • 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 os. 6,914,128, 5,780,597, and 5,81 1,523 ; Roland E. Kontermann and Stefan Dubel (editors), Antibody Engineering. Vol. I & H. (2010) 2 nd ed., Springer; Antony S.
  • antibodies directed to an EGFR fusion molecule can be obtained commercially from Abeam, Santa Cruz Biotechnology, Abgent, R&D Systems, Novus Biologicals, etc.
  • Human antibodies directed to an EGFR fusion molecule can be useful antibody therapeutics for use in humans.
  • an antibody or binding fragment thereof is directed against SEQ ID NOS: I, 3, 5, 7, 9, 1 1 , 13, 15, or 17.
  • Antisense nucleotide sequences include, but are not limited to: morpholinos, 2'-0-methyl
  • polynucleotides DNA , RNA and the like.
  • 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 R A 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 covalentlv 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.
  • 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-nucleo tides.
  • One or both strands of the siRN A can also comprise a 3' overhang.
  • a 3' overhang refers to at least one unpaired nucleotide extending from the 3'-end of a duplexed RNA strand.
  • 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.
  • each strand of the siRNA can comprise 3' overhangs of dithymidylic acid ("TT") or diuridylic acid ("uu").
  • 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 Harmon et ai., and in U.S. Patent No, 7, 148,342 to Tolentino et ai., the entire disclosures of which are herein incorporated by reference.
  • EGFR fusion molecule inhibitor can be a small molecule that binds to an EGFR 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 an EGFR fusion protein can be identified via in silico screening or high-through-put 0 1 ! P ) screening of combinatorial libraries according to methods established in the art (e.g., see Potyrailo et al., (201 1 ) ACS Comb Sei.
  • EGFR inhibitors include, but are not limited to:
  • a structure of an EGFR fusion molecule inhibitor useful for the invention includes, but is not limited to the inhibitor KHS101 ,
  • 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 glioblastoma multiforme, breast cancer, lung cancer, prostate cancer, or colorectal carcinoma.
  • the method comprises detecting the presence of an EGFR fusion molecule in a sample obtained from a subject.
  • the sample is incubated with an agent that binds to an EFGR fusion molecule, such as an antibody, a probe, a nucleic acid primer, and the like.
  • the method comprises administering to the subject an effective amount of an EGFR fusion molecule inhibitor, wherein the inhibitor decreases the size of the solid tumor.
  • the invention also provides a method for treating or preventing a gene-fusion associated cancer in a subject, such as, but not limited to, glioblastoma multiforme, breast cancer, lung cancer, prostate cancer, or colorectal carcinoma.
  • the method comprises detecting the presence of an EGFR fusion molecule in a sample obtained from a subject, the presence of the fusion being indicative of a gene-fusion associated cancer, and, administering to the subject in need a therapeutic treatment against a gene-fusion associated cancer.
  • the sample is incubated with an agent that binds to an EFGR fusion molecule, such as an antibody, a probe, a nucleic acid primer, and the like.
  • the invention also provides a method for decreasing in a subject in need thereof the expression level or activity of a fusion protein comprising the tyrosine kinase domain of an EGFR protein fused to a polypeptide that constitutively activates the tyrosine kinase domain of the EGFR protein.
  • the method comprises obtaining a biological sample from the subject.
  • the sample is incubated with an agent that binds to an EGFR tusion molecule, such as an antibody, a probe, a nucleic acid primer, and the like.
  • 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 tusion protein of the invention.
  • the method further comprises determining the fusion protein expression level or activity.
  • 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.
  • the fusion protein is an EGFR-PSPH fusion protein, an EGFR-CAND fusion protein, or an EGFR-SEPT fusion protein.
  • the administering step in each of the claimed methods can comprise a drug administration, such as EGFR fusion molecule inhibitor (for example, a pharmaceutical composition comprising an antibody that specifically binds to an EGFR-SEPT fusion protein, an EGFR-PSPH fusion protein, an EGFR-CAND fusion protein, or a fragment thereof; a small molecule that specifically binds to an EGFR protein; an antisense RNA or antisense DNA that decreases expression of an EGFR-SEPT fusion protein, an EGFR-PSPH fusion protein, an EGFR-CAND fusion; a. siRNA that specifically targets an EGFR-SEPT fusion gene, an EGFR-PSPH fusion gene, or an EGFR-CAND).
  • EGFR fusion molecule inhibitor for example, a pharmaceutical composition comprising an antibody that specifically binds to an EGFR-SEPT fusion protein, an EGFR-PSPH fusion protein, an EGFR-CAND fusion protein, or a fragment thereof;
  • the therapeutic molecule to be administered comprises a polypeptide of an EGFR 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 NOS: 1, 3, 5, 7, 9, 1 1 , 13, 15, or 17, and exhibits the function of decreasing expression of such a protein, thus treating a gene fusion-associated cancer.
  • administration of the therapeutic molecule decreases the size of the solid tumor associated with glioblastoma multiforme, breast cancer, lung cancer, prostate cancer, or colorectal carcinoma.
  • glioblastoma multiforme glioblastoma multiforme
  • breast cancer breast cancer
  • lung cancer lung cancer
  • prostate cancer or colorectal carcinoma
  • the therapeutic molecule to be administered comprises an siRNA directed to a human nucleic acid sequence comprising an EGFR fusion molecule.
  • the siRNA is directed to any one of SEQ ID NOS: 2, 4, 8, 10, 14, or 16.
  • the therapeutic molecule to be administered comprises an antibody or binding fragment thereof, that is directed against SEQ ID NOS: 1, 3, 5, 7, 9, 1 1, 13, 15, or 17.
  • the therapeutic molecule to be administered comprises a small molecule that specifically binds to an EGFR protein, such as AZD4547, NVP-BGJ398, PD173074, NF449, TK1258, BIBF-1120, BMS-582664, AZD-2171, TSU68, AB1010, AP24534, E-7080, or LY2874455.
  • an EGFR protein such as AZD4547, NVP-BGJ398, PD173074, NF449, TK1258, BIBF-1120, BMS-582664, AZD-2171, TSU68, AB1010, AP24534, E-7080, or LY2874455.
  • An EGFR fusion molecule for example, a fusion between EGFR and SEPT, PSPH, or CAND, can be determined at the level of the D A, RNA, or polypeptide.
  • detection can be determined by performing an oligonucleotide ligation assay, a. confirmation based assay, a hybridization assay, a sequencing assay, an aliele-specific amplification assay, a microsequencing assay, a melting curve analysis, a denaturing high performance liquid chrom tography (DHPLC) assay (for example, see Jones et al, (2.000) Hum Genet., 106(6):663-8), or a combination thereof.
  • DPLC denaturing high performance liquid chrom tography
  • the detection is performed by sequencing all or part of an EGFR fusion molecule (e.g., EGFR-SEPT fusion (such as an EGFR-SEPT 14 fusion), EGFR-CAND fusion (such as an EGFR-CAND 1 fusion), EGFR-PSPH), or by selective hybridization or amplification of all or part of an EGFR fusion molecule (e.g., EGFR-SEPT fusion (such as an EGFR-SEPT 14 fusion), EGFR-CAND fusion (such as an EGFR-CAND 1 fusion), EGFR-PSPH)).
  • an EGFR fusion molecule e.g., EGFR-SEPT fusion (such as an EGFR-SEPT 14 fusion), EGFR-CAND fusion (such as an EGFR-CAND 1 fusion), EGFR-PSPH).
  • EGFR fusion molecule specific amplification e.g., EGFR-SEPT (such as an EGFR-SEPT 14), EGFR-CAND (such as an EGFR-CAND 1), EGFR-PSPH nucleic acid specific amplification
  • EGFR-SEPT such as an EGFR-SEPT 14
  • EGFR-CAND such as an EGFR-CAND 1
  • EGFR-PSPH nucleic acid specific amplification can be carried out before the fusion identification step.
  • the invention provides for a method of detecting a chromosomal alteration in a subject afflicted with a gene-fusion associated cancer.
  • the gene-fusion associated cancer comprises glioblastoma multiforme, breast cancer, lung cancer, prostate cancer, or colorectal carcinoma.
  • the chromosomal alteration is an in- frame fused transcript described herein, for example an EGFR fusion molecule.
  • the alteration in a chromosome region occupied by an EGFR fusion molecule 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 an EGFR fusion molecule, for example, a nucleic acid encoding an EGFR-SEPT fusion (such as an EGFR-SEPT 14 fusion), an EGFR-CAND fusion (such as an EGFR-CAND 1 fusion), or an EGFR-PSPH fusion, with altered function, stability, targeting or structure.
  • an EGFR-SEPT fusion such as an EGFR-SEPT 14 fusion
  • an EGFR-CAND fusion such as an EGFR-CAND 1 fusion
  • an EGFR-PSPH fusion an EGFR-PSPH fusion
  • the present invention provides a method for treating a gene-fusion associated cancer in a subject in need thereof.
  • the method comprises obtaining a sample from the subject to determine the level of expression of an EGFR fusion molecule in the subject.
  • the sample is incubated with an agent that binds to an EGFR fusion molecule, such as an antibody, a probe, a nucleic acid primer, and the like.
  • the detection or determination comprises nucleic acid sequencing, selective hybridization, selective amplification, gene expression analysis, or a combination thereof.
  • the detection or determin tion comprises protein expression analysis, for example by western blot analysis, ELISA, or other antibody detection methods.
  • the detection or determination comprises nucleic acid sequencing, selective hybridization, selective amplification, gene expression analysis, or a combination thereof.
  • the detection or determination comprises protein expression analysis, for example by western blot, analysis, ELISA, or other antibody detection methods.
  • the method further comprises assessing whether to administer an EGFR fusion molecule inhibitor based on the expression pattern of the subject.
  • the method comprises administering an EGFR 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.
  • t e method can comprise detecting the presence or expression of an EGFR fusion molecule, such as a nucleic acid encoding an EGFR-SEPT fusion (such as an EGFR-SEPT 14 fusion), an EGFR-CAND fusion (such as an EGFR- CAND1 fusion), or an EGFR-PSPH fusion.
  • 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).
  • the detecting comprises using a northern blot; real time PGR and primers directed to SEQ ID NOS: 2, 4, 8, 10, 14, or 16; a
  • Hybridization detection methods are based on the formation of specific hybrids between complementary nucleic acid sequences that serve to detect nucleic acid sequence aiteration(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.
  • the probe according to the invention can comprise a nucleic acid directed to SEQ ID NOS: 2, 4, 8, 10, 14, or 16.
  • 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 an EGFR 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 an EGFR fusion molecule.
  • the sequence of the probes can be derived from the sequences of the EGFR 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. [00181] 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
  • Sequencing can be carried out using techniques well known in the art, using automatic sequencers. The sequencing can be performed on the complete EGFR fusion molecule or on specific domains thereof.
  • Amplification is based on the formation of specific hybrids between complementary nucleic acid sequences that serve to initiate nucleic acid
  • Amplification can be performed according to various techniques known in the art, such as by polymerase chain reaction (PGR), 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 PGR, aliele-specific PGR, or PGR based single-strand conformational polymorphism (SSCP). Amplification usually requires the use of specific nucleic acid primers, to initiate the reaction.
  • PGR polymerase chain reaction
  • LCR ligase chain reaction
  • SDA strand displacement amplification
  • NASBA nucleic acid sequence based amplification
  • Useful techniques in the art encompass real-time PGR, aliele-specific PGR, or PGR based single-strand conformational polymorphism (SSCP).
  • SSCP single-strand conformational polymorphism
  • nucleic acid primers useful for amplifying sequences corresponding to an EGFR fusion molecule are able to specifically hybridize with a portion of the gene locus that flanks a target region of the locus.
  • amplification comprises using forward and reverse PGR primers directed to SEQ ID NOS: 2, 4, 8, 10, 14, or 16.
  • the presence of an EGFR fusion molecule corresponds to a subject with a gene fusion-associated cancer.
  • amplification can comprise using forward and reverse PGR primers comprising nucleotide sequences of SEQ ID NOS: 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29.
  • the invention provides for a nucleic acid primer, wherein the primer can be complementary to and hybridize specifically to a portion of an EGFR fusion molecule, such as a nucleic acid (e.g., DNA or RNA), in certain subjects having a gene fusion-associated cancer.
  • a nucleic acid e.g., DNA or RNA
  • the gene-fusion associated cancer comprises glioblastoma multiforme, breast cancer, lung cancer, prostate cancer, or colorectal carcinoma.
  • 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 an EGFR fusion molecule, e.g. a nucleic acid encoding an EGFR-SEPT fusion (such as an EGFR-SEPT 14 fusion), an EGFR-CAND fusion (such as an EGFR-CAND 1 fusion), or an EGFR-PSPH fusion. Perfect complementarity is useful to ensure high specificity; however, certain mismatch can be tolerated.
  • a nucleic acid encoding an EGFR fusion molecule or expression of an EGFR 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.
  • the sample is contacted with an antibody specific for a polypeptide encoded by an EGFR fusion molecule and fee formation of an immune complex is subsequently determined.
  • an immune complex Various methods for detecting an immune complex can be used, such as ELISA, radioimmunoassays (RIA) and immuno-enzymatic assays ( ⁇ ).
  • 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 an EGFR fusion molecule can be an antibody that selectively binds such a polypeptide. In one embodiment, fee antibody is raised against a polypeptide encoded by an EGFR fusion molecule or an epitope-containing fragment thereof.
  • the kit can comprise nucleic acid primers that specifically hybridize to and can prime a polymerase reaction from an EGFR fusion molecule comprising SEQ ID NOS: 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or a combination thereof.
  • primers can be used to detect an EGFR fusion molecule, such as a primer comprising SEQ ID NOS: 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29; or a combination thereof.
  • primers used for the screening of EGFR fusion molecules can comprise nucleic acid primers that specifically hybridize to and can prime a polymerase reaction from an EGFR fusion molecule comprising SEQ ID NOS: 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or a combination thereof.
  • 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 an EGFR 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.
  • the sample is contacted with reagents, such as probes, primers, or ligands, in order to assess the presence of an EGFR fusion molecule. Contacting can be performed in any suitable device, such as a plate, tube, well, or glass.
  • 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.
  • herpesvimses including HSV and EBV (Margolskee (1992) Curr Top
  • Non-limiting examples of in vivo gene transfer techniques include transfectkm 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-iiposome mediated transfection (Dzau et al, (1993) Trends in Biotechnology 11 :205-210), incorporated entirely by reference).
  • viral e.g., retroviral
  • viral coat protein-iiposome mediated transfection Dzau et al, (1993) Trends in Biotechnology 11 :205-210
  • 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.
  • 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 Mgdicd Application supra, vol. 2, pp.
  • compositions suitable for administration for example the inhibitor and a pharmaceutically acceptable carrier
  • 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.
  • 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 in vention are characterized as being at least sterile and pyrogen- free. These pharmaceutical formulations include formulations for human and veterinary use.
  • a pharmaceutical composition containing EGFR fission 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 an EGFR fusion molecule, or a variant thereof, or antagonists of an EGFR 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.
  • 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 ethylenediaminetetraacetic 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.
  • the carrier can be a solvent or dispersion medium containing, for example, water, erhanol, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions can contain any of the following ingredients, or compounds of a similar nature: a binder such as microciystaliine 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.
  • a binder such as microciystaliine 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
  • Systemic administration can also be by transmucosai or transdermal means.
  • penetrants appropriate to the hairier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosai administration, detergents, bile salts, and fusidic acid derivati ves.
  • Transmucosai administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.
  • the effective amount of the administered EGFR fusion molecule inhibitor is at least about 0.0001 ⁇ /3 ⁇ 43 ⁇ 4 body weight, at least about 0.00025 ⁇ gJkg body weight, at least about 0.0005 .ug/kg body weight, at least about 0.00075 ,ug/kg body weight, at least about 0.001 ⁇ g!
  • Examplejj The integrated landscape of driver genomic alterations in glioblastoma
  • ⁇ -catenin in mesenchymal glioma cells reprogrammed them towards a neuronal cell fate.
  • Recurrent translocations were also identified that fuse in-frame the coding sequence of EGFR to several partners in 7.6% of tumors, with EGFR-Septin- 14 scoring as the most frequent functional gene fusion in human glioblastoma.
  • EGFR fusions enhance proliferation and motility of glioma cells and confer sensitivity to EGFR inhibition in glioblastoma xenografts.
  • GBM Glioblastoma
  • BCOR a chromosome X-linked gene, encodes for a componen t of the nuclear co-repressor complex that is essential for normal development of the neuroectoderm and stem cell functions 1 1"1 3 .
  • BCOR mutations have recently been described in retinoblastoma and medulloblastoma, thus indicating that loss-of- function mutations in BCOR are common genetic events in neuroectodermal tumors 14 ' 13 .
  • Herc2 The gene coding for the Hect biquitin ligase Herc2 is localized on chromosome 15ql 3 and is deleted and mutated in 15.1 % and 2.2% of GBM cases, respectively (Table 4). This gene has been implicated in severe neurodevelopmental syndromes. Moreover, protein substrates of Herc2 are crucial factors in genome stability and DNA damage-repair, two cell functions frequently disrupted in cancer" '2 '.
  • LZTR-1 mutants To address the potential function of LZTR-1 mutants, a homology model of LZTR-1 was built based in part on the crystal structures of the MATH-BTB-BACK protein SPOP 26 , the BTB-BACK-Kelch proteins KLHL3 and KLHL11 27 , and the Kelch domain of Keapl 28 (Fig. 2b).
  • the second BTB-BACK region of LZTR- 1 binds Cul3 because of the presence of a ⁇ - ⁇ - ⁇ motif in this BTB domain, foilowed by a 3 - Box/BACK region (Fig. 9) 26 .
  • the preceding BTB-BACK region also participates in Cul3 binding.
  • CTNND2 is the gene expressed at the highest levels in the normal brain (Table 6).
  • CTNND2 codes for -catenin, a member of the i 20 subfamily of catenins that is expressed almost exclusively in the nervous system where it is crucial for neurite elongation, dendritic morphogenesis and synaptic plasticity 31 "' " '.
  • Germ-line hemizygous loss of CTNND2 severely impairs cognitive function and underlies some forms of mental retardation" 4 ' " ' 3 .
  • CTNND2 shows a pronounced clustering of mutations in GBM.
  • immunoflurescence staining on brain tumor tissue microarrays were performed as previously described 5*1 '. Immunofluorescence microscopy was performed on cells fixed with 4% para-formaldehyde (PFA) in phosphate buffer. Cells were permeabilized using 0.2% Triton X 100. Antibodies and concentrations used in immunofluorescence staining are:
  • hPSPH-RT-REV 1 5'- TGCCTGATCACATTTCCTCCA-3 ' (SEQ ID NO: 35);
  • genomic CAND1-FW1 5'- GCAATAGCA AAACAGGAAGATGTC-3 ' (SEQ ID NO: 44);
  • Erlotinib was administered at lOOmg/Kg orally daily for 10 days.
  • Lapatinib was administered at 75mg/Kg orally twice per day for 20 days.
  • Response to treatment was assessed by delay in tumor growth and tumor regression.
  • Growth delay expressed as T-C, is defined as the difference in days between the median time required for tumors in treated and control animals to reach a volume five times greater than that measured at the start of the treatment.
  • Tumor regression is defined as a decrease in tumor volume over two successive measurements.
  • Statistical analysis was performed using a SAS statistical analysis program, the Wilcoxon rank order test for growth delay, and Fisher's exact test for tumor regression as previously described.
  • ChimeraScan a tool for identifying chimeric transcription in sequencing data, Bioinformatics 27, 2903-2904, doi: 10.1093/bioinformatics btr467 (201 1 ).
  • Glioblastoma remains one of the mos t challenging forms of cancer to treat.
  • This example discusses a computational platform that integrates the analysis of copy number variations and somatic mutations and unravels the landscape of in-frame gene fusions in glioblastoma. Mutations were found with loss of heterozygosity of LZTR-1, an adaptor of Cul3-containing E3 Hgase complexes. Mutations and deletions disrupt LZTR-1 function, which restrains self-renewal and growth of glioma spheres retaining stem cell features. Loss- of-function mutations of CTNND2 target a neural-specific gene and are associated with transformation of glioma cells along the very aggressive mesenchymal phenotype.
  • GBM Glioblastoma.
  • MutComFocal is an algorithm designed to rank genes by an integrated recurrence, focality and mutation score (see Methods). This strategy was applied to 139 GBM and matched normal DNA analyzed by whole-exome sequencing to identify somatic mutations and 469 GBM analyzed by the Affymetrix SNP6.0 platform to identify CNVs.
  • LRP proteins are highly expressed in the neuroepithelium and are essential for forebrain morphogenesis in mouse and humans 1 '' 18 .
  • the tumor suppressor function of LRP proteins in GBM may relate to the ability to promote chemosensitivity and control in the Sonic hedgehog signaling pathway, which is implicated in cancer initiating cells in GBM 19" ' '1 .
  • Localized on chromosome 15ql3, the Hect ubiquiiin ligase Herc2 gene is deleted and mutated in 15.1 % and 2.2% of GBM cases, respectively (Tabic 4).
  • Herc2 has been implicated in severe neurodevelopmental syndromes and Herc2 substrates regulate genome stability and D A damage-repair 22 ' 2"5 .
  • LZTR- 1 A gene that received one of the highest Del-Mut score by MutComFocal is LZTR- 1 (Fig. lc, Table 4).
  • the LZTR-l coding region had non-synonymous mutations in 4.4%, and the LZTR-l locus (human chromosome 22ql l) was deleted in 22.4% of GBM.
  • LZTR-l codes for a protein with a characteristic Kelch-BTB-BACK-BTB-BACK domain architecture (FIGS. 2C, 8, 9) and is expressed in normal brain (Table 6).
  • the LZTR- 1 gene is highly conserved in metazoans. Although it was initially proposed that LZTR-l functions as a transcriptional regulator, this role was not confirmed in follow-up studies 24 .
  • Cul3 ubiquitin 3iga.se complexes in which the BTB-BACK region binds to the N-terminal domain of Cul3, while a ligand binding domain, often a Kelch 6-bladed ⁇ -propeller motif, binds to substrates targeted for ubiquitylation" 3 .
  • a ligand binding domain often a Kelch 6-bladed ⁇ -propeller motif
  • FIG. 15 shows that Cul3 immunoprecipitates contain LZTR-l, indicating thai LZTR-l is an adaptor in Cul3 ubiquitin ligase complexes.
  • LZTR-i mutations identified in GBM Five of seven LZTR-i mutations identified in GBM are located within the Keich domain and target highly conserved amino acids (Fig. 2b, FIG. 2C, FIG. 8). Interestingly, the concentration of LZTR-1 mutations in the Kelch domain reflects a similar pattern of mutations in the Kelch-coding region of KLHL3, recently identified in families with hypertension and electrolytic abnormalities " ' 0 ' 31 .
  • the R398G and G248R mutations localize to the b-c loop of the Kelch domain, in a region predicted to provide the substrate-binding surface 21 '.
  • the W105R mutation targets a highly conserved anchor residue in the Kelch repeats and the T288I mutation disrupts a buried residue conserved in LZTR-1 (Fig. 2b, FIG. 2C, FIG. 8). Both mutations are expected to perturb folding of the Kelch domain.
  • the E353STOP mutation is expected to produce a misfolded Kelch domain besides removing the C-terminal BTB-BACK regions.
  • Cullin adaptors are short-lived proteins that undergo auto-ubiquityiation and destruction by the same Cullin complexes that direct substrate ubiquitylation 32"' .
  • impaired ubiquitin ligase activity of the LZTR-1 -CuB complex should result in accumulation of mutant LZTR-i proteins.
  • Each of the three LZTR-1 mutants predicted to compromise integrity of the BTB-BACK domains accumulated at higher levels than wild-type LZTR-1 in transient transfection assays (FIG. 16B).
  • the steady state and half-life of the LZTR-1 R810W mutant protein were markedly increased, in the absence of changes of the mutant mR A (FIG. 16C-D).
  • the R810W mutation compromised protein degradation
  • LZTR-1 Differential gene expression pattern of GBM harboring mutations was examined and deletions of LZTR-1 or normal LZTR-1 revealed thai tumors with genetic inaetivation of LZTR-1 were enriched for genes associated with glioma sphere growth and proliferation 3"1 (FIG. 17.4).
  • Introduction of LZTR-1 in three independent GBM-derived sphere cultures resulted in strong inhibition of glioma sphere formation and expression of glioma stem cell markers (FIG. 17B-E).
  • LZTR-1 also decreased the size of tumor spheres, induced a flat and adherent phenotype and reduced proteins associated with cell cycle progression (cyclin A, PLKl, pl07, FIG. 17D-E).
  • CTNND2 is expressed at the highest levels in normal brain ( Table 6).
  • CTNND2 codes for ⁇ -catenin, a member of the l20 subfamily of catenins expressed almost exclusively in the nervous system where it is crucial for neurite elongation, dendritic morphogenesis and synaptic plasticity 36"38 .
  • Germ-line hemizygous loss of CTNND2 impairs cognitive functions and underlies some forms of mental retardation 39 ' 40 .
  • CTNND2 shows pronounced clustering of mutations in GBM.
  • the observed spectrum of mutations includes four mutations in the armadillo-coding domain and one in the region coding for the N-terminal coiled-coil domain (FIG. 10A), the two most relevant functional domains of ⁇ -caienin.
  • Each mutation targets highly conserved residues with probably (K629Q, A776T, S881L, D999E) and possibly (A71T) damaging consequences 4 ' .
  • GBM harbors focal genomic losses of CTNND2, and deletions correlate with loss of
  • mesenchymal markers was analyzed 4j .
  • introduction of ⁇ -catenin in sphere culture #48 strongly reduced mesenchymal proteins smooth muscle actin (SMA), collagen- 5A1 (ColSAl) and FBN, as measured by quantitative immunofluorescence (FIGS. 19A-B). It also induced ⁇ -tubulin more than eight-fold (FIGS, 1.9C-D).
  • Time course analysis showed the highest degree of ⁇ -tubulin-positive neurite extension at 4-6 days post-transduction followed by progressive depletion of neuronal Tike cells from culture (FIG. 19D).
  • 5- catenin impacts self-renewal and growth of glioma spheres in vitro and their ability to grow- as tumor masses in vivo were examined.
  • RNA-seq data was analyzed from a total of 185 GBM samples (161 primary GBM plus 24 short-term glioma sphere cultures freshly isolated from patients carrying primary GBM).
  • the analysis of RNA-seq led to the discovery of 92 candidate rearrangements giving rise to in-frame fusion transcripts (Table 7).
  • Table 7 Besides previously reported FGFR3-TACC3 fusions events, the most frequent recurrent in-frame fusions involved EGFR in 7.6% of samples (14/185, 3.8%- 11.3% CI).
  • SEPT14 recurrent partners
  • SPH 3/185, 1.6%) as the 3' gene segment in the fusion. All EGFR-SEPT14 and two of three EGFR-PSPH gene fusions occurred within amplified regions of the fusion genes (FIG, 24).
  • NTRK1 neurotrophic tyrosine kinase receptor 1 gene
  • EXomeFuse an algorithm that reconstructs genomic fusions from whole-exome data, EGFR-SEPT14 and NRTKJ fusions result from recurrent chromosomal translocations and the corresponding genomic breakpoints were reconstructed (Table 12).
  • FIGS. 4A-B The sequence of the PGR products spanning the fusion breakpoint validated all three types of recurrent in-frame fusion predictions (EGFR-SEPTJ 4, EGFR-PSPH and NRTK1 fusions, FIGS. 4, 11, 12).
  • FIGS. 4A-B the prediction and cDNA sequence validation is shown respectively, for one tumor harboring an EGFR-SEPT14 fusion (TCGA- 27-1837).
  • the amplified cDNA contained an open reading frame for a 1,041 amino-acid protein resulting from the fusion of EGFR residues 1-982 with SEPT 14 residues 373-432 (FIG. 4C).
  • EGFR.-Septin 14 fusions involves EGFR at the N-termimis, providing a receptor tyrosine kinase domain fused to a coiled-coii domain from Septi l.4.
  • Ex on-specific R A-seq expression in TCGA-27-1837 demonstrated that EGFR and SEPT 14 exons implicated in the fusion are highly expressed compared with mRNA sequences not included in the fusion event (FIG. 13),
  • the genomic breakpoint was mapped to chromosome 7 (#55,268,937 for EGFR and # 55,870,909 for SEPT14, genome build GRCh37/hgl9) within EGFR exon 25 and SEPT 14 intron 9, creating a transcript in which the 5' EGFR exon 24 is spliced to the 3 ' SEPT 14 exon 10 (FIG. 4D).
  • the fused EGFR-PSPH cDNA and predicted fusion protein in sample TCGA -06-5408 involves the same EGFR N -terminal region implicated in the EGFR-SEPT14 with PSPH roviding a carboxy-terminal portion of 35 amino acids (FIG. 11).
  • a fusion in which the EGFR-TK region is the 3 ' partner is the CAND1-EGFR fusion in the glioma sphere culture #16 (FIG. 14).
  • Each fusion transcript includes the region of the EGFR mRNA coding for the TK domain (Table 7).
  • RT- PCR and genomic PGR followed by Sanger sequencing of GBM TCGA-06-5411 validated the NFASC-NTRKI fusions in which the predicted fusion protein includes the TK domain of the high-affinity NGF receptor (TrkA) fused downstream to the immunoglobulin-like region of the cell adhesion and ankyrin-binding region of neurofascin (FIG. 12).
  • TrkA high-affinity NGF receptor
  • EGFR-SEPT14 might constitutively activate signaling events downstream of EGFR.
  • Differential gene expression analysis identified a set of 9 genes up- regulated in EGFR-SEPT] 4 tumors compared with EGFRvIII-poskkQ GBM (FIG. 26). These genes broadly relate to inflammatory/immune response, and some code for chemokmes (CXCL9, 10, 11) that have been associated with aggressive glioma phenotypes 46 .
  • LZTR-1 mutations targeting highly conserved residues in the Kelch domain (W105, G248, T288) and in the second BTB-BACK domain (R810) are recurrent events in other tumor types 45 .
  • understanding the nature of substrates of LZTR-1 -CuB ubiquttin ligase activity will provide important insights into the pathogenesis of multiple cancer types.
  • LZTR -1 genetic alterations in GBM is underscored by concurrent targeting of LZTR-1 by mutations and deletions that supports a two-hits mechanism of tumor suppressor gene inactivation as well as the impact of mutations targeting the BTB-BACK domains on CuB binding and/or protein stability, and their ability to release glioma cells from the restraining activity of the wild -type protein on self-renewal,
  • SA VI statistical algorithm far variant frequency identification
  • the frequencies of variant alleles were estimated in 139 paired tumor and normal whole-exome samples from TCGA using the SAVI pipeline 30 .
  • the algorithm estimates the frequency of variant alleles by constructing an empirical Bayesiaii prior for those frequencies, using data from the whole sample, and obtains a posterior distribution and high credibility intervals for each allele 50 .
  • the prior and posterior are distributed over a discrete set of frequencies with a precision of 1 % and are connected by a modified binomial likelihood, which allows for some error rate. More precisely, a prior distribution p(j) of the frequency /and a.
  • the sequencing data at a. particular allele is a random experiment producing a string of (the total depth at the allele) bits with n Ts (the variant depth at the allele). Assuming a binomial likelihood of the data and allowing for bits being misread because of random errors, the posterior probability P(J) of the frequency /is where C is a normalization constant. For a particular allele, the value ofE is determined by the quality of t he nucleotides sequenced at that position as specified by their Phred scores.
  • the SAVI pipeline takes as input the reads produced by the sequencing technology, filters out low-quality reads and maps the rest onto a human reference genome. After mapping, a Bayesian prior for the distribution of allele frequencies for each sample is constructed by an iterative posterior update procedure starting with a uniform prior. To genotype the sample, the posterior high-credibility intervals were used for the f equency of the alleles at each genomic location. Alternatively, combining the Bayesian priors from different samples, posterior high-credibility intervals were obtained for the difference between the samples of the frequencies of each allele. Finally, the statistically significant differences between the tumor and normal samples are reported as somatic variants. The results are shown in Table 1.
  • Table 3 shows the candidate genes ranked by the number of somatic
  • nonsynonymous mutations A robust fit of the ratio of nonsynonymous to synonymous mutations was generated with a bisquare weighting function. The excess of nonsynonymous alterations was estimated using a Poisson distribution with a mean equal to the product of the ratio from the robust fit and the number of synonymous mutations.
  • Genes in highly polymorphic genomic regions were filtered out based on an independent cohort of normal samples. The list of these regions includes families of genes known to generate false positives in somatic predictions (for example, s e ///.. !. A ' 7?Z ' and OR gene families).
  • MutComFocal Key cancer genes are often amplified or deleted in chromosomal regions containing many other genes. Point mutations and gene fusions, conversely, provide more specific information about which genes may be implicated in the oncogenic process.
  • MutComFocal was developed, a Bayesian approach that assigns a driver score to each gene by integrating point mutations and CNV data, from 469 GBMs (Affymetrix SNP6.0). In general, MutComFocal uses three different strategies. First, the focality component of the score is inversely proportional to the size of the genomic lesion to which a gene belongs and thus prioritizes more focal genomic lesions.
  • the recurrence component of the MutComFocal score is inversely proportional to the total number of genes altered in a sample, which prioritizes samples with a smaller number of altered genes.
  • the mutation component of the score is inversely proportional to the total n umber of genes mutated in a sample, which achieves the twofold goal of prioritizing mutated genes on one hand and prioritizing samples with a smaller number of mutations on the other.
  • the amplification/mutation score is defined as the product of the two
  • amplification/mutation and deletion/mutation scores are normalized to 1 , and for each score, genes are divided into tiers iterative! ⁇ ' so that the top 2 X remaining genes are included in the next tier, where H is the entropy of the scores of the remaining genes normalized to 1.
  • genes are assigned to being either deleted/mutated or amplified/mutated, and genes in the top tiers are grouped into contiguous regions. The top genes in each region are considered manually and selected for further functional validation.
  • the recurrence and focality scores can be interpreted as the posterior probabilities that a gene is dri ving the selection of the disease under two different priors, one global and one local in nature.
  • the recurrence score is higher if a gene participates in many samples that do not have too many altered genes, whereas the focality score is higher if the gene participates in many focal lesions.
  • the directionality of the copy number alteration informs the probable behavior of the candidate gene as an oncogene or tumor suppressor, respectively.
  • RNA-seq hwinformatics analysis 161 RNA-seq GBM tumor samples were analyzed from TCGA, a public repository' containing large-scale genome sequencing of different cancers, plus 24 patient-derived GSCs. Nine GSC samples reported in previous studies were kept in our analysis to evaluate recurrence 9 . The samples were analyzed using the ChimeraScan* 1 algorithm to detect a list of gene fusion candidates. Briefly, ChimeraScan detects those reads that discordantly align to different transcripts of the same reference (split inserts). These reads provide an initial set of putative fusion candidates. The algorithm then realigns the initially unmapped reads to the putative fusion candidates and detects those reads that align across the junction boundary (split reads). These reads provide the genomic coordinates of the breakpoint,
  • RNA-seq analysis detected a total of 39,329 putative gene fusion events.
  • the Pegasus annotation pipeline http://sourceforge.net/projects/pegasus-rus/
  • Pegasus reconstructs the entire fusion sequence on the basis of the genomic fusion breakpoint coordinates and gene annotations.
  • Pegasus also annotates the reading frame of the resulting fusion sequences as either in frame or a frame shift.
  • Pegasus detects the protein domains that are either conserved or lost in the new chimeric event by predicting the amino acid sequence and automatically querying the UniProt web service.
  • EXome-Fuse a new gene fusion discovery pipeline that is designed particularly to analyze whole-exome data.
  • EXome-Fuse was applied to the corresponding whole-exome sequencing data deposited in TCGA.
  • This algorithm can be divided into three stages: split-insert identification, split-read identification and virtual reference alignment. Mapping against the human genome reference hg!8 with BWA, all split inserts are first identified to compile a preliminary list of fusion candidates.
  • Targeted exon sequencing All protein-coding exons for the 24 genes of interest were sequenced using genomic DNA extracted from frozen tumors and matched blood. Five- hundred nanograms of DMA from each sample were sheared to an average size of 150 bp in a Covaris instrument for 360 s (duty cycle, 10%; intensity, 5; cycles per burst, 200). Bar-coded libraries were prepared using the Kapa High-Throughput Library Preparation Kit Standard (Kapa Biosvstems). Libraries were amplified using the KAPA HiFi Library Amplification kit (Kapa Biosvstems) (eight cycles). Libraries were quantified using Qubit Fiuorimetric Quantitation (Invitrogen), and the quality and size was assessed using an Agilent
  • Bioanalyzer An equimolar pool of the four bar-coded libraries (300 ng each) was created, and 1 ,200 ng was input to exon capture using one reaction tube of the custom Nimbiegen SeqCap EZ (Roche) with custom probes targeting the coding exons of the 38 genes.
  • Capture by hybridization was performed according to the manufacturer's protocols with the following modifications: 1 nmol of a pool of blocker oligonucleotides (complementary to the bar-coded adapters) was used, and post-capture PCR amplification was done using the KAPA HiFi Library Amplification kit, instead of the Phusion High-Fidelity PCR Master Mix with HF Buffer Kit, in a 60 ⁇ volume, as the Kapa HiFi kit greatly reduced or eliminated the bias against GC-rich regions.
  • the pooled capture library was quantified by Qubit (Invitrogen) and Bioanalyzer (Agilent) and sequenced in on an Illumina MiSeq sequencer using the 2 x 150 paired-end cycle protocol. Reads were aligned to the hg!9 build of the human genome using BWA with duplicate removal using SAMtools as implemented by lilumina MiSeq Reporter. Variant detection was performed using GATK UnifiedGenofyper. Somatic mutations were identified for paired samples using SomaticSniper and filtered for frequency of less than 3% in normal samples and over 3% in tumor samples.
  • MutComFocal combines SNV and CNV data to identify genes driving oncogenesis, it does not explicitly determine whether amplified or deleted genes are enriched for SNVs within the same sample. Deletions and SNVs of a gene within the same sample might indicate a two-hit model of a tumor suppressor. Alternati vely, amplifications and gain- of-function mutations of an oncogene within the sample might further promote oncogenesis. For each MutComFocal candidate gene, the number of TCGA samples was determined with both amplification and SNVs, amplification alone, SNVs alone or neither. The
  • RNA sequencing can determine whether the mutant or wild-type allele is expressed.
  • VCFtools54 was applied to the TCGA BAM RNA-seq files produced by TopHat, which produces the depth of reads calling the reference (R.) and variant (V) allele.
  • a measure of relative expression of the variant allele is then V/(V ⁇ R).
  • the binomial P value of observing more than V out of V + R reads was calculated, assuming that it is equally probable for a read to call the variant or reference.
  • the binomial P values of each mutation were then pooled using the Stouffer's Z-score method to calculate the combined P value per gene.
  • segmented S P array data was downloaded from TCGA and calculated the log2 ratio between the tumor and normal copy numbers. This was plotted along the chromosomal neighborhood of EGFR, SEPT 14 and SPH (chr7:55,000,0()()-56,500,000).
  • GSEA To determine the biological impact oi LZTRI mutations, GSEA 55 was used, which is an analytical tool that harnesses expression data to nominate gene sets enriched for a particular phenotype. Having identified TCGA samples with LZTRI SNVs, GSEA was applied to the TCGA expression data. Samples were first compared with LZTRI SNVs against those with wild-type LZTRI (excluding LZTRI deletions). To assess statistical significance, the data set was randomized by permuting gene sets 500 times and considered only gene sets with an FDR q ⁇ 0,05.
  • Modeling of LZTRI Structural templates for the kelch and BTB-BACK regions of human LZTRI were identified with HBpred 50 .
  • An initial three-dimensional model was generated with the I-TASSER server 5 '.
  • the CUL3 N -terminal domain was docked onto the model by superposing the LHL3 B1B"BACK /CUL3 NlD crystal structure 2 ' onto the second LZTR 1 BTB-BACK. domain.
  • the model does not include higher quaternary structure, although many BTB domains, and many kelch domains, are known to self associate 25 .
  • BACK domains are the shorter, atypical form of the domain and consist of two helical hairpin motifs, as in SPOP 2 *' 58 , and not the four-hairpin motif seen in most BTB- BAC -kelch proteins 28, 58 .
  • the model from the kelch domain predicts an unusual 1 +3 velcro arrangement 39 , with the -terminal region contributing strand d of blade 1 and the C-terminal region contributing strands a, b and c of the same blade, although an alternative 2+2 velcro model cannot be ruled out.
  • U87 cells were obtained from ATCC. SNB19, U87 and HE -293T cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Growth rates were determined by plating cells in six-well plates at 3 d after infection with the lentiviras indicated in the figure legends. The number of viable cells was determined by Trypan blue exclusion in triplicate cultures obtained from triplicate independent infections. For the wound assay testing migration, confluent cells were scratched with a pipette tip and cultured in 0.25% FBS. After 16 li, images were taken using the Olympus 1X70 connected to a digital camera. Images were processed using the lmageJ64 software. The area of the ceil- free wound was assessed in triplicate samples. Experiments were repeated twice.
  • FBS fetal bovine serum
  • GBM-derived primary cultures were grown in DMEM:F12 medium containing N2 and B27 supplements and human recombinant FGF-2 and EGF (50 ng-'mi each;
  • Spheres were dissociated into single cells and plated in low-attachment 96-well plates in 0.2 ml of medium containing growth factors (EGF and FGF-2), except for the EGFR-transduced cells, which were cultured in the absence of EGF.
  • EGF and FGF-2 growth factors
  • Each treatment group was seeded in triplicate. Absolute viable cell counts were determined by Trypan blue exclusion and counted on a hemocytometer. EGF stimulation of EGFR-transduced primary glioma cells was performed in cells deprived of growth factors for 48 h. Cells were collected at the indicated times and processed for protein blot analysis.
  • Fiag-CUL3 was immunoprecipitated from transfected ⁇ -293 ⁇ cells with Flag-M2 beads (Sigma.) using R1PA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxychoiate (DOC), 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride fPMSF), 10 mM NaF. 0.5 M asOV 4 (sodium orthovanadate) and Complete Protease Inhibitor Cocktail, Roche). Binding was performed in 200 mM NaCl plus 0.5% NP-40 for 2 h at 4 °C.
  • R1PA buffer 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxychoiate (DOC), 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride fPMSF
  • 10 mM NaF 0.5 M
  • the lentiviral expression vectors pLOC-GFP and pLOC-CTNND2 were purchased from Open Biosystems. Full-length EGFR-SEPT14 cDNA was amplified from tumor sample TCGA-27--1837. Wild-type EGFR, EGFRvlll and EGFR-SEPT14 cDNAs were cloned into the pLOC vector.
  • pCDNA-MYC-Hist-LZTRl was a kind gift 24 .
  • pCDNA-Flag- CUL3 was a gift.
  • fusion transcripts The validation of fusion transcripts was performed using both genomic PGR and RT-PCR with forward and reverse primer combinations designed within the margins of the paired- end read sequences detected by RNA-seq. Expressed fusion transcript variants were subjected to direct sequencing to confirm the sequence and translation frame.
  • the primers used for the screening of gene fusions are detailed in Table 17.
  • the primers used for genomic detection of gene fusions are listed in Table 18.
  • Semiquantitative RT-PCR to detect exogenous wild-type MYC-LZTRl and mutant p.ArgSOl Trp LZTRI was performed using the primers listed in Table 19.
  • Growth delay expressed as a T-C value, is defined as the difference in days between the median time required for tumors in treated and control animals to reach a. volume five times greater than that measured at the start of the treatment. Tumor regression is defined as a decrease in tumor volume over two successive measurements. Statistical analysis was performed using a SAS statistical analysis program, the Wilcoxon rank-order test for growth delay and Fisher's exact test for tumor regression.
  • RNA sequencing of twenty-four human GBM sphere cultures in this study were deposited under the dbGaP study accession phs000505.v2.pl, RNA and DNA sequencing of TCGA GBM samples was also analyzed from the dbGaP study accession phs000178.vl .pl .

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Epidemiology (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Cell Biology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • General Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Toxicology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • General Physics & Mathematics (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
PCT/US2014/026351 2013-03-15 2014-03-13 Fusion proteins and methods thereof Ceased WO2014151734A1 (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
CA2907152A CA2907152A1 (en) 2013-03-15 2014-03-13 Fusion proteins and methods thereof
EP14769771.8A EP2968551B1 (en) 2013-03-15 2014-03-13 Fusion proteins and methods thereof
AU2014236947A AU2014236947A1 (en) 2013-03-15 2014-03-13 Fusion proteins and methods thereof
JP2016502111A JP2016515508A (ja) 2013-03-15 2014-03-13 融合タンパク質及びその方法
KR1020157029166A KR20150129847A (ko) 2013-03-15 2014-03-13 융합 단백질 및 이들의 방법
US14/853,568 US10208296B2 (en) 2013-03-15 2015-09-14 Fusion proteins and methods thereof
US16/246,167 US11505788B2 (en) 2013-03-15 2019-01-11 Fusion proteins and methods thereof
US18/046,131 US20230303985A1 (en) 2013-03-15 2022-10-12 Fusion proteins and methods thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201361793086P 2013-03-15 2013-03-15
US61/793,086 2013-03-15

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/853,568 Continuation US10208296B2 (en) 2013-03-15 2015-09-14 Fusion proteins and methods thereof

Publications (1)

Publication Number Publication Date
WO2014151734A1 true WO2014151734A1 (en) 2014-09-25

Family

ID=51581025

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2014/026351 Ceased WO2014151734A1 (en) 2013-03-15 2014-03-13 Fusion proteins and methods thereof

Country Status (7)

Country Link
US (3) US10208296B2 (enExample)
EP (1) EP2968551B1 (enExample)
JP (1) JP2016515508A (enExample)
KR (1) KR20150129847A (enExample)
AU (1) AU2014236947A1 (enExample)
CA (1) CA2907152A1 (enExample)
WO (1) WO2014151734A1 (enExample)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016105517A1 (en) * 2014-12-23 2016-06-30 The Trustees Of Columbia University In The City Of New York Fusion proteins and methods thereof
US10208296B2 (en) 2013-03-15 2019-02-19 The Trustees Of Columbia University In The City Of New York Fusion proteins and methods thereof
CN109810184A (zh) * 2019-01-17 2019-05-28 武汉明德生物科技股份有限公司 人nf155抗原、人nf155抗体检测试剂盒及其制备方法与应用
EP3555274A4 (en) * 2016-12-13 2020-08-05 University of Pittsburgh - of The Commonwealth System of Higher Education METHODS OF TREATMENT OF CANCERS CONTAINING FUSION GENES
EP4017873A4 (en) * 2019-08-23 2023-12-20 Duke University COMPOSITIONS AND METHODS FOR THE TREATMENT OF PATHOLOGICAL PAIN AND ITCH
US11920138B2 (en) 2012-07-24 2024-03-05 The Trustees Of Columbia University In The City Of New York Fusion proteins and methods thereof
WO2024103032A3 (en) * 2022-11-11 2024-06-20 The Trustees Of Columbia University In The City Of New York Lztr1 mutant tumors and methods thereof
WO2025019568A1 (en) * 2023-07-17 2025-01-23 The Penn State Research Foundation Improving immune cell-based therapies against solid tumors with optogenetically engineered septin proteins

Families Citing this family (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8450322B2 (en) 2008-09-22 2013-05-28 Array Biopharma Inc. Substituted imidazo[1,2b]pyridazine compounds as Trk kinase inhibitors
TR201807039T4 (tr) 2008-10-22 2018-06-21 Array Biopharma Inc Trk kinaz inhibitörleri olarak sübstitüe edilmiş pirazolo[1,5-]pirimidin bileşikleri.
AR077468A1 (es) 2009-07-09 2011-08-31 Array Biopharma Inc Compuestos de pirazolo (1,5 -a) pirimidina sustituidos como inhibidores de trk- quinasa
ES2628418T3 (es) 2010-05-20 2017-08-02 Array Biopharma, Inc. Compuestos macrocíclicos como inhibidores de la TRK cinasa
US20160053301A1 (en) 2014-08-22 2016-02-25 Clearfork Bioscience, Inc. Methods for quantitative genetic analysis of cell free dna
PT3699181T (pt) 2014-11-16 2023-04-05 Array Biopharma Inc Forma cristalina de hidrogenossulfato de (s)-n-(5-((r)-2-(2,5-difluorofenil)-pirrolidin-1-il)-pirazolo[1,5-a]pirimidin-3-il)-3-hidroxipirrolidina-1-carboxamida
TN2018000138A1 (en) 2015-10-26 2019-10-04 Array Biopharma Inc Point mutations in trk inhibitor-resistant cancer and methods relating to the same
CN109414442B (zh) 2016-04-04 2024-03-29 洛克索肿瘤学股份有限公司 一种化合物的液体制剂
US10045991B2 (en) 2016-04-04 2018-08-14 Loxo Oncology, Inc. Methods of treating pediatric cancers
DK3800189T3 (da) 2016-05-18 2023-07-31 Loxo Oncology Inc Fremstilling af (s)-n-(5-((r)-2-(2,5-difluorphenyl)pyrrolidin-1-yl)pyrazolo[1,5-a]pyrimidin-3-yl)-3-hydroxypyrrolidin-1-carboxamid
JOP20190092A1 (ar) 2016-10-26 2019-04-25 Array Biopharma Inc عملية لتحضير مركبات بيرازولو[1، 5-a]بيريميدين وأملاح منها
AU2017258901A1 (en) * 2016-12-30 2018-07-19 Allarity Therapeutics Europe ApS Methods for predicting drug responsiveness in cancer patients
JOP20190213A1 (ar) 2017-03-16 2019-09-16 Array Biopharma Inc مركبات حلقية ضخمة كمثبطات لكيناز ros1
WO2019083594A1 (en) * 2017-08-21 2019-05-02 The General Hospital Corporation COMPOSITIONS AND METHODS FOR CLASSIFYING TUMORS WITH INSTABILITY OF MICROSATELLITES
US11110177B2 (en) * 2017-11-10 2021-09-07 The Regents Of The University Of Michigan ASH1L degraders and methods of treatment therewith
KR102277471B1 (ko) * 2019-10-22 2021-07-14 주식회사 지니스 '혈관내 혈전' 용해제
KR102362115B1 (ko) * 2019-10-22 2022-02-14 주식회사 지니스 '혈관내 혈전' 용해제
EP4114979A4 (en) * 2020-03-04 2025-08-27 Found Medicine Inc BCOR REDESIGNS AND THEIR USES
WO2024064679A1 (en) * 2022-09-20 2024-03-28 Foundation Medicine, Inc. Methods and systems for functional status assignment of genomic variants
CN115312119B (zh) * 2022-10-09 2023-04-07 之江实验室 基于蛋白质三维结构图像鉴定蛋白质结构域的方法及系统

Citations (42)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4921475A (en) 1983-08-18 1990-05-01 Drug Delivery Systems Inc. Transdermal drug patch with microtubes
US5008110A (en) 1988-11-10 1991-04-16 The Procter & Gamble Company Storage-stable transdermal patch
US5087240A (en) 1983-08-18 1992-02-11 Drug Delivery Systems Inc. Transdermal drug patch with conductive fibers
US5088977A (en) 1988-12-21 1992-02-18 Drug Delivery Systems Inc. Electrical transdermal drug applicator with counteractor and method of drug delivery
US5164189A (en) 1989-12-04 1992-11-17 G. D. Searle & Co. Single layer transdermal drug administration system
US5163899A (en) 1987-03-20 1992-11-17 Drug Delivery Systems Inc. Transdermal drug delivery system
US5252479A (en) 1991-11-08 1993-10-12 Research Corporation Technologies, Inc. Safe vector for gene therapy
US5254346A (en) 1988-02-23 1993-10-19 Tucker Mark J Occlusive body for administering a physiologically active substance
US5328470A (en) 1989-03-31 1994-07-12 The Regents Of The University Of Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor
US5332213A (en) 1992-01-29 1994-07-26 Franz Volkl Gmbh & Co. Ski Und Tennis Sport-Artkihelfabrik Kg. Ball-game racket, particularly a tennis racket
US5336168A (en) 1987-05-28 1994-08-09 Drug Delivery Systems Inc. Pulsating transdermal drug delivery system
US5352456A (en) 1991-10-10 1994-10-04 Cygnus Therapeutic Systems Device for administering drug transdermally which provides an initial pulse of drug
US5407713A (en) 1991-12-18 1995-04-18 Minnesota Mining And Manufacturing Company Multilayered barrier structures
US5747469A (en) 1991-03-06 1998-05-05 Board Of Regents, The University Of Texas System Methods and compositions comprising DNA damaging agents and p53
US5780597A (en) 1989-12-22 1998-07-14 Hoffmann-La Roche Inc. Monoclonal antibodies to cytotoxic lymphocyte maturation factor
US5811523A (en) 1988-11-10 1998-09-22 Trinchieri; Giorgio Antibodies to natural killer stimulatory factor
WO1999032619A1 (en) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Genetic inhibition by double-stranded rna
WO1999035159A1 (en) * 1998-01-08 1999-07-15 Brigham & Women's Hospital, Inc. Lymphoma/leukemia oncogene, oncoprotein and methods of use
WO2000001846A2 (en) 1998-07-03 2000-01-13 Devgen N.V. Characterisation of gene function using double stranded rna inhibition
US6017524A (en) 1991-03-06 2000-01-25 Board Of Regents, The University Of Texas System Inhibiting the growth p53 deficient tumor cells by administering the p53 gene
WO2000044914A1 (en) 1999-01-28 2000-08-03 Medical College Of Georgia Research Institute, Inc. Composition and method for in vivo and in vitro attenuation of gene expression using double stranded rna
WO2000044895A1 (de) 1999-01-30 2000-08-03 Roland Kreutzer Verfahren und medikament zur hemmung der expression eines vorgegebenen gens
US6143290A (en) 1992-10-13 2000-11-07 The Board Of Regents, University Of Texas System Tumor regression by adenovirus expression of wild-type p53
WO2001029058A1 (en) 1999-10-15 2001-04-26 University Of Massachusetts Rna interference pathway genes as tools for targeted genetic interference
WO2001032840A2 (en) 1999-11-03 2001-05-10 Amanda Jane Reynolds Hair transplantation
US20020077313A1 (en) 1995-11-30 2002-06-20 Board Of Regents, The University Of Texas System Methods and compositions for the diagnosis and treatment of cancer
US20020173478A1 (en) 2000-11-14 2002-11-21 The Trustees Of The University Of Pennsylvania Post-transcriptional gene silencing by RNAi in mammalian cells
US6509154B1 (en) 1997-08-04 2003-01-21 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) Product comprising at least a double stranded RNA combined with at least an antiviral agent
US20030027783A1 (en) 1999-11-19 2003-02-06 Magdalena Zernicka-Goetz Inhibiting gene expression with dsRNA
US6914128B1 (en) 1999-03-25 2005-07-05 Abbott Gmbh & Co. Kg Human antibodies that bind human IL-12 and methods for producing
US7148342B2 (en) 2002-07-24 2006-12-12 The Trustees Of The University Of Pennyslvania Compositions and methods for sirna inhibition of angiogenesis
US7294504B1 (en) 2001-12-27 2007-11-13 Allele Biotechnology & Pharmaceuticals, Inc. Methods and compositions for DNA mediated gene silencing
WO2008075068A2 (en) 2006-12-21 2008-06-26 Astrazeneca Ab Acylaminopyrazoles as fgfr inhibitors
US7419661B2 (en) 1997-04-30 2008-09-02 The Centre Of Excellence For Life Sciences Limited Dermal sheath tissue in wound healing
US20080312248A1 (en) 2005-12-21 2008-12-18 Novartis Ag Pyrimidinyl Aryl Urea Derivatives Being Fgf Inhibitors
US20090318480A1 (en) * 2006-09-18 2009-12-24 Boehringer Ingelheim International Gmbh Method for treating cancer harboring egfr mutations
US20100120678A1 (en) * 2006-10-13 2010-05-13 The Regents Of The University Of California The Office Of The President Novel inhibitors of the egfr kinase targeting the asymmetric activating dimer interface
WO2010129509A1 (en) 2009-05-07 2010-11-11 Eli Lilly And Company Vinyl indazolyl compounds
US20110023143A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of neurodevelopmental genes in animals
US8071559B2 (en) 2005-05-02 2011-12-06 Cold Spring Harbor Laboratory Compositions and methods for cancer diagnosis and treatment

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BRPI0814542A2 (pt) 2007-07-12 2014-09-30 Tragara Pharmaceuticals Inc Métodos e composições para o tratamento de câncer, tumores e desordens relacionadas a tumores
US20150203589A1 (en) 2012-07-24 2015-07-23 The Trustees Of Columbia University In The City Of New York Fusion proteins and methods thereof
CN104797936B (zh) 2012-07-24 2017-11-24 纽约市哥伦比亚大学理事会 融合蛋白及其方法
WO2014151734A1 (en) 2013-03-15 2014-09-25 The Trustees Of Columbia University In The City Of New York Fusion proteins and methods thereof
WO2016105517A1 (en) 2014-12-23 2016-06-30 The Trustees Of Columbia University In The City Of New York Fusion proteins and methods thereof

Patent Citations (48)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4921475A (en) 1983-08-18 1990-05-01 Drug Delivery Systems Inc. Transdermal drug patch with microtubes
US5087240A (en) 1983-08-18 1992-02-11 Drug Delivery Systems Inc. Transdermal drug patch with conductive fibers
US4683202B1 (enExample) 1985-03-28 1990-11-27 Cetus Corp
US4683202A (en) 1985-03-28 1987-07-28 Cetus Corporation Process for amplifying nucleic acid sequences
US4683195B1 (enExample) 1986-01-30 1990-11-27 Cetus Corp
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US5163899A (en) 1987-03-20 1992-11-17 Drug Delivery Systems Inc. Transdermal drug delivery system
US5336168A (en) 1987-05-28 1994-08-09 Drug Delivery Systems Inc. Pulsating transdermal drug delivery system
US5254346A (en) 1988-02-23 1993-10-19 Tucker Mark J Occlusive body for administering a physiologically active substance
US5008110A (en) 1988-11-10 1991-04-16 The Procter & Gamble Company Storage-stable transdermal patch
US5811523A (en) 1988-11-10 1998-09-22 Trinchieri; Giorgio Antibodies to natural killer stimulatory factor
US5088977A (en) 1988-12-21 1992-02-18 Drug Delivery Systems Inc. Electrical transdermal drug applicator with counteractor and method of drug delivery
US5328470A (en) 1989-03-31 1994-07-12 The Regents Of The University Of Michigan Treatment of diseases by site-specific instillation of cells or site-specific transformation of cells and kits therefor
US5164189A (en) 1989-12-04 1992-11-17 G. D. Searle & Co. Single layer transdermal drug administration system
US5290561A (en) 1989-12-04 1994-03-01 G. D. Searle & Co. Single layer transdermal drug administration system
US5780597A (en) 1989-12-22 1998-07-14 Hoffmann-La Roche Inc. Monoclonal antibodies to cytotoxic lymphocyte maturation factor
US6017524A (en) 1991-03-06 2000-01-25 Board Of Regents, The University Of Texas System Inhibiting the growth p53 deficient tumor cells by administering the p53 gene
US5747469A (en) 1991-03-06 1998-05-05 Board Of Regents, The University Of Texas System Methods and compositions comprising DNA damaging agents and p53
US5352456A (en) 1991-10-10 1994-10-04 Cygnus Therapeutic Systems Device for administering drug transdermally which provides an initial pulse of drug
US5252479A (en) 1991-11-08 1993-10-12 Research Corporation Technologies, Inc. Safe vector for gene therapy
US5407713A (en) 1991-12-18 1995-04-18 Minnesota Mining And Manufacturing Company Multilayered barrier structures
US5332213A (en) 1992-01-29 1994-07-26 Franz Volkl Gmbh & Co. Ski Und Tennis Sport-Artkihelfabrik Kg. Ball-game racket, particularly a tennis racket
US6410010B1 (en) 1992-10-13 2002-06-25 Board Of Regents, The University Of Texas System Recombinant P53 adenovirus compositions
US6143290A (en) 1992-10-13 2000-11-07 The Board Of Regents, University Of Texas System Tumor regression by adenovirus expression of wild-type p53
US6511847B1 (en) 1992-10-13 2003-01-28 Board Of Regents, The University Of Texas System Recombinant p53 adenovirus methods and compositions
US20020077313A1 (en) 1995-11-30 2002-06-20 Board Of Regents, The University Of Texas System Methods and compositions for the diagnosis and treatment of cancer
US7419661B2 (en) 1997-04-30 2008-09-02 The Centre Of Excellence For Life Sciences Limited Dermal sheath tissue in wound healing
US6509154B1 (en) 1997-08-04 2003-01-21 Societe De Conseils De Recherches Et D'applications Scientifiques (S.C.R.A.S.) Product comprising at least a double stranded RNA combined with at least an antiviral agent
WO1999032619A1 (en) 1997-12-23 1999-07-01 The Carnegie Institution Of Washington Genetic inhibition by double-stranded rna
WO1999035159A1 (en) * 1998-01-08 1999-07-15 Brigham & Women's Hospital, Inc. Lymphoma/leukemia oncogene, oncoprotein and methods of use
WO2000001846A2 (en) 1998-07-03 2000-01-13 Devgen N.V. Characterisation of gene function using double stranded rna inhibition
WO2000044914A1 (en) 1999-01-28 2000-08-03 Medical College Of Georgia Research Institute, Inc. Composition and method for in vivo and in vitro attenuation of gene expression using double stranded rna
WO2000044895A1 (de) 1999-01-30 2000-08-03 Roland Kreutzer Verfahren und medikament zur hemmung der expression eines vorgegebenen gens
US6914128B1 (en) 1999-03-25 2005-07-05 Abbott Gmbh & Co. Kg Human antibodies that bind human IL-12 and methods for producing
WO2001029058A1 (en) 1999-10-15 2001-04-26 University Of Massachusetts Rna interference pathway genes as tools for targeted genetic interference
WO2001032840A2 (en) 1999-11-03 2001-05-10 Amanda Jane Reynolds Hair transplantation
US20030027783A1 (en) 1999-11-19 2003-02-06 Magdalena Zernicka-Goetz Inhibiting gene expression with dsRNA
US20020173478A1 (en) 2000-11-14 2002-11-21 The Trustees Of The University Of Pennsylvania Post-transcriptional gene silencing by RNAi in mammalian cells
US7294504B1 (en) 2001-12-27 2007-11-13 Allele Biotechnology & Pharmaceuticals, Inc. Methods and compositions for DNA mediated gene silencing
US7422896B1 (en) 2001-12-27 2008-09-09 Allele Biotechnology & Pharmaceuticals, Inc. Compositions for DNA mediated gene silencing
US7148342B2 (en) 2002-07-24 2006-12-12 The Trustees Of The University Of Pennyslvania Compositions and methods for sirna inhibition of angiogenesis
US8071559B2 (en) 2005-05-02 2011-12-06 Cold Spring Harbor Laboratory Compositions and methods for cancer diagnosis and treatment
US20080312248A1 (en) 2005-12-21 2008-12-18 Novartis Ag Pyrimidinyl Aryl Urea Derivatives Being Fgf Inhibitors
US20090318480A1 (en) * 2006-09-18 2009-12-24 Boehringer Ingelheim International Gmbh Method for treating cancer harboring egfr mutations
US20100120678A1 (en) * 2006-10-13 2010-05-13 The Regents Of The University Of California The Office Of The President Novel inhibitors of the egfr kinase targeting the asymmetric activating dimer interface
WO2008075068A2 (en) 2006-12-21 2008-06-26 Astrazeneca Ab Acylaminopyrazoles as fgfr inhibitors
US20110023143A1 (en) * 2008-12-04 2011-01-27 Sigma-Aldrich Co. Genomic editing of neurodevelopmental genes in animals
WO2010129509A1 (en) 2009-05-07 2010-11-11 Eli Lilly And Company Vinyl indazolyl compounds

Non-Patent Citations (124)

* Cited by examiner, † Cited by third party
Title
"Animal Cell Culture: A Practical Approach", 1992, OXFORD UNIVERSITY PRESS
"Antibody Engineering", vol. I, II, 2010, SPRINGER
"Antibody Engineering: Methods and Protocols (Methods in Molecular Biology)", 2004, HUMANA PRESS
"Controlled Drug Bioavailability, Drug Product Design and Performance", 1984, WILEY
"Culture Of Animal Cells", 1987, ALAN R. LISS, INC.
"Current Protocols In Molecular Biology", 2001, JOHN WILEY & SONS, INC.
"DNA Cloning", vol. I, II, 1985
"Gene Transfer Vectors For Mammalian Cells", 1987, COLD SPRING HARBOR LABORATORY
"Genebank", Database accession no. NP_ 005219
"Genebank", Database accession no. NP_958439
"Genebank", Database accession no. NP_958440
"Genebank", Database accession no. NP_958441
"Handbook Of Experimental Immunology", vol. I-IV, 1986
"Immobilized Cells and Enzymes", 1986, IRL PRESS
"Immunochemical Methods In Cell And Molecular Biology", 1987, ACADEMIC PRESS
"Manipulating the Mouse Embryo", 1986, COLD SPRING HARBOR LABORATORY PRESS
"Medical Applications of Controlled Release", 1974, CRC PRES.
"Methods In Enzymology", ACADEMIC PRESS, INC.
"Methods In Enzymology", vol. 154, 155
"Molecular Cloning: A Laboratory Manual", vol. 1-3, 1989, COLD SPRING HARBOR LABORATORY
"Nucleic Acid Hybridization", 1984
"Oligonucleotide Synthesis", 1984
"PCR Protocols, A Guide To Methods And Applications", 1990, ACADEMIC PRESS
"PCR Strategies", 1995, ACADEMIC PRESS, INC.
"Remmington's Pharmaceutical Sciences", 2000, MEADE PUBLISHING CO.
"Theory and Nucleic Acid Preparation", 1993, ELSEVIER, article "Laboratory Techniques In Biochemistry And Molecular Biology: Hybridization With Nucleic Acid Probes"
"Therapeutic Antibodies: Methods and Protocols (Methods in Molecular Biology)", 2009, HUMANA PRESS
"Transcription and Translation", 1984
ALTARAS ET AL., ADV BIOCHEM ENG BIOTECHNOL., vol. 99, 2005, pages 193 - 260
ANDERSON ET AL., SCIENCE, vol. 256, 1992, pages 808 - 813
ANDERSON, NATURE, vol. 392, no. 6679, 1998, pages 25
ATHERTON ET AL.: "Solid Phase Peptide Synthesis: a Practical Approach", 1989, IRL PRESS
B. PERBAL, A PRACTICAL GUIDE TO MOLECULAR CLONING, 1984
BANDYOPADHYAY; TEMIN, MOL CELL BIOL., vol. 4, no. 4, 1984, pages 749 - 54
BARRINGER, GENE, vol. 89, 1990, pages 117
BASS, NATURE, vol. 411, 2001, pages 428 - 429
BERGER, METHODS ENZYMOL., vol. 152, 1987, pages 307 - 316
BERKNER, BIOTECHNIQUES, vol. 6, no. 7, 1988, pages 616 - 29
BERKNER, CURR TOP MICROBIOL IMMUNOL., vol. 158, 1992, pages 39 - 66
BREAKEFIELD; GELLER, MOL NEUROBIOL., vol. 1, no. 4, 1987, pages 339 - 71
BROWER, NATURE BIOTECHNOLOGY, vol. 16, 1998, pages 1304 - 1305
BUCHSCHACHER; PANGANIBAN, J VIROL., vol. 66, no. 5, 1992, pages 2731 - 9
BUCHWALD ET AL., SURGERY, vol. 88, 1980, pages 507
BURG, MOL. CELL. PROBES, vol. 10, 1996, pages 257 - 271
CHEN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 91, 1994, pages 3054 - 3057
CLEVELAND WL ET AL., J IMMUNOL METHODS, vol. 56, no. 2, 1983, pages 221 - 234
DALLAS ET AL., MED. SCI. MONIT., vol. 12, no. 4, 2006, pages RA67 - 74
DURING ET AL., ANN. NEUROL., vol. 25, 1989, pages 351
DZAU ET AL., TRENDS IN BIOTECHNOLOGY, vol. 11, 1993, pages 205 - 210
ELBASHIR ET AL., NATURE, vol. 411, 2001, pages 494 498
ELIYAHU ET AL., MOLECULES, vol. 10, no. l, 2005, pages 34 - 64
F. M. AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 1989, JOHN WILEY & SONS
FINK ET AL., HUM GENE THER., vol. 3, no. 1, 1992, pages 11 - 9
FRATTINI ET AL., NATURE GENETICS, vol. 45, no. 10, 2013, pages 1141 - 49
FREESE ET AL., BIOCHEM PHARMACOL., vol. 40, no. 10, 1990, pages 2189 - 99
FRIEDMANN, SCIENCE, vol. 244, 1989, pages 1275 - 1281
GAVINE ET AL., CANCER RES, vol. 72, no. 8, 2012, pages 2045 - 56
GOODSON, MEDICAL APPLICATIONS OF CONTROLLED RELEASE, vol. 2, 1984, pages 115 - 138
GORZIGLIA; KAPIKIAN, J VIROL., vol. 66, no. 7, 1992, pages 4407 - 12
GUAGNANO ET AL., J. MED. CHEM., vol. 54, 2011, pages 7066 - 7083
GUATELLI, PNAS, vol. 87, 1990, pages 1874
HELSETH ET AL., J VIROL., vol. 64, no. 12, 1990, pages 6314 - 8
HERWEIJER ET AL., GENE THER., vol. 14, no. 2, 2007, pages 99 - 107
HOFFMAN, A. S., ADV DRUG DELIV REV., vol. 43, 2002, pages 3 - 12
HOFFMAN, A. S., ANN NY ACAD SCI, vol. 944, 2001, pages 62 - 73
HOWARD ET AL., J. NEUROSURG., vol. 71, 1989, pages 105
HUDSON, CURR. OPIN. BIOTECHNOL., vol. 9, 1998, pages 395 - 402
ISAKA ET AL., EXPERT OPIN DRUG DELIV., vol. 4, no. 5, 2007, pages 561 - 71
J. SAMBROOK ET AL.: "Molecular Cloning, A Laboratory Manual", 2001, COLD SPRING HARBOR PRESS
JAGER ET AL., CURR GENE THER., vol. 7, no. 4, 2007, pages 272 - 83
JENSEN ET AL., ANN MED., vol. 39, no. 2, 2007, pages 108 - 15
JHOTI, ERNST SOBERING FOUND SYMP PROC., vol. 3, 2007, pages 169 - 85
JOHNSON ET AL., BRAIN RES MOL BRAIN RES., vol. 12, no. 1-3, 1992, pages 95 - 102
JONES ET AL., HUM GENET., vol. 106, no. 6, 2000, pages 663 - 8
KALOTA ET AL.: "Handb. Exp. Pharmacol.", vol. 173, 2006, pages: 173 - 96
KIKUCHI ET AL., J DERMATOL SCI., vol. 50, no. 2, 2008, pages 87 - 98
KREJCI, JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 285, no. 27, 2010, pages 20644 - 20653
KWOH, PNAS, vol. 86, 1989, pages 1173
LANDEGREN, SCIENCE, vol. 241, 1988, pages 1077
LANGER, SCIENCE, vol. 249, 1990, pages 1527 - 1533
LEE ET AL., BIOMATERIALS, vol. 25, 2004, pages 2461 - 2466
LEVY ET AL., SCIENCE, vol. 228, 1985, pages 190
LIU, J ET AL.: "NEDD8 Modification Of CUL1 Dissociates P120cand1. An Inhibitor Of CUL1-SKP1 Binding And SCF Ligases", MOLECULAR CELL ., vol. 10, December 2002 (2002-12-01), pages 1511 - 1518, XP055279592 *
LUTZELBURGER ET AL.: "Handb. Exp. Pharmacol.", vol. 173, 2006, pages: 243 - 59
MADZAK ET AL., J GEN VIROL., vol. 73, 1992, pages 1533 - 6
MANN; BALTIMORE, J VIROL., vol. 54, no. 2, 1985, pages 401 - 7
MARGOLSKEE, CURR TOP MICROBIOL IMMUNOL., vol. 158, 1992, pages 67 - 95
MAYNARD ET AL., ANN. REV. BIOMED. ENG., vol. 2, 2000, pages 339 - 76
MCMNAUS; SHARP, NAT REV GENETICS, vol. 3, 2002, pages 737 - 47
MENSCH ET AL., JPHARM SCI., vol. 98, no. 12, 2009, pages 4429 - 68
MILLER ET AL., J VIROL., vol. 55, no. 3, 1985, pages 521 - 6
MILLER ET AL., J VIROL., vol. 62, no. 11, 1988, pages 4337 - 45
MILLER ET AL., MOL CELL BIOL., vol. 12, no. 7, 1992, pages 3262 - 72
MILLER, NATURE, vol. 357, 1992, pages 455 - 460
MOHAMMADI ET AL., EMBO J., vol. 17, 1998, pages 5896 - 5904
MOSS, CURR OPIN BIOTECHNOL., vol. 3, no. 5, 1992, pages 518 - 22
MUZYCZKA, CURR TOP MICROBIOL IMMUNOL., vol. 158, 1992, pages 97 - 129
OHI ET AL., GENE., vol. 89, no. 2, 1990, pages 279 - 82
PAGE ET AL., J VIROL., vol. 64, no. 11, 1990, pages 5270 - 6
PEPPAS ET AL., ADV MATER., vol. 18, 2006, pages 1345 - 60
PETERSON, E ET AL.: "Characterization Of A SEPT9 Interacting Protein, SEPT14, A Novel Testis-Specific Septin.", MAMM GENOME., vol. 18, 26 June 2007 (2007-06-26), pages 796 - 807, XP019561875 *
PETROPOULOS ET AL., J VIROL., vol. 66, no. 6, 1992, pages 3391 - 7
POTYRAILO ET AL., ACS COMB SCI., vol. 13, no. 6, 2011, pages 579 - 633
QUANTIN ET AL., PROC NATL ACAD SCI USA., vol. 89, no. 7, 1992, pages 2581 - 4
RANGER; PEPPAS, J. MACROMOL. SCI. REV. MACROMOL. CHEM., vol. 23, 1983, pages 61
ROSENFELD ET AL., CELL., vol. 68, no. 1, 1992, pages 143 - 55
SAMBROOK; FRITSCH; MANIATIS: "Molecular Cloning A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAUDEK ET AL., N. ENGL. J. MED., vol. 321, 1989, pages 574
SCHNUR, CURR OPIN DRUG DISCOV DEVEL., vol. 11, no. 3, 2008, pages 375 - 80
SEFTON, BIOMED. ENG., vol. 14, 1987, pages 201
SEN; BLAU, FASEB J., vol. 20, 2006, pages 1293 - 99
SHIMADA ET AL., J CLIN INVEST., vol. 88, no. 3, 1991, pages 1043 - 7
SHINODA, T ET AL.: "Septin 14 Is Involved In Cortical Neuronal Migration Via Interaction With Septin 4.", MOLECULAR BIOLOGY OF THE CELL, vol. 21, 15 April 2010 (2010-04-15), pages 1324 - 1334, XP055279598 *
SMITH, J. CLIN. MICROBIOL., vol. 35, 1997, pages 1477 - 1491
SOOKNANAN, BIOTECHNOLOGY, vol. 13, 1995, pages 563 - 564
SORGE ET AL., MOL CELL BIOL., vol. 4, no. 9, 1984, pages 1730 - 7
STRATFORD-PERRICAUDET ET AL., HUM GENE THER., vol. 1, no. 3, 1990, pages 241 - 56
VERMA, SCIENTIFIC AMERICAN, 1990, pages 68 - 84
WAEHLER ET AL., NAT REV GENET., vol. 8, no. 8, 2007, pages 573 - 87
WERNER ET AL., BRIEF FUNCT. GENOMIC PROTEOMIC, vol. 5, no. l, 2006, pages 32 - 6
WILKINSON ET AL., NUCLEIC ACIDS RES., vol. 20, no. 9, 1992, pages 2233 - 9
WU, GENOMICS, vol. 4, 1989, pages 560
WURDAK ET AL., PNAS, vol. 107, no. 38, 2010, pages 16542 - 47
ZHAO ET AL., MOL CANCER THER., vol. 11, 2011, pages 2200 - 10

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11920138B2 (en) 2012-07-24 2024-03-05 The Trustees Of Columbia University In The City Of New York Fusion proteins and methods thereof
US12123005B2 (en) 2012-07-24 2024-10-22 The Trustees Of Columbia University In The City Of New York Fusion proteins and methods thereof
US12006503B2 (en) 2012-07-24 2024-06-11 The Trustees Of Columbia University In The City Of New York Fusion proteins and methods thereof
US11505788B2 (en) 2013-03-15 2022-11-22 The Trustees Of Columbia University In The City Of New York Fusion proteins and methods thereof
US10208296B2 (en) 2013-03-15 2019-02-19 The Trustees Of Columbia University In The City Of New York Fusion proteins and methods thereof
WO2016105517A1 (en) * 2014-12-23 2016-06-30 The Trustees Of Columbia University In The City Of New York Fusion proteins and methods thereof
US11384400B2 (en) 2016-12-13 2022-07-12 University of Pittsburgh—of the Commonwealth System of Higher Education Methods of treating cancers containing fusion genes
EP3555274A4 (en) * 2016-12-13 2020-08-05 University of Pittsburgh - of The Commonwealth System of Higher Education METHODS OF TREATMENT OF CANCERS CONTAINING FUSION GENES
CN109810184B (zh) * 2019-01-17 2022-07-12 武汉明德生物科技股份有限公司 人nf155抗原、人nf155抗体检测试剂盒及其制备方法与应用
CN109810184A (zh) * 2019-01-17 2019-05-28 武汉明德生物科技股份有限公司 人nf155抗原、人nf155抗体检测试剂盒及其制备方法与应用
EP4017873A4 (en) * 2019-08-23 2023-12-20 Duke University COMPOSITIONS AND METHODS FOR THE TREATMENT OF PATHOLOGICAL PAIN AND ITCH
WO2024103032A3 (en) * 2022-11-11 2024-06-20 The Trustees Of Columbia University In The City Of New York Lztr1 mutant tumors and methods thereof
WO2025019568A1 (en) * 2023-07-17 2025-01-23 The Penn State Research Foundation Improving immune cell-based therapies against solid tumors with optogenetically engineered septin proteins

Also Published As

Publication number Publication date
AU2014236947A1 (en) 2015-09-03
CA2907152A1 (en) 2014-09-25
US20230303985A1 (en) 2023-09-28
US10208296B2 (en) 2019-02-19
US20160108380A1 (en) 2016-04-21
EP2968551A1 (en) 2016-01-20
KR20150129847A (ko) 2015-11-20
US20190203188A1 (en) 2019-07-04
US11505788B2 (en) 2022-11-22
EP2968551A4 (en) 2016-08-24
EP2968551B1 (en) 2021-05-05
JP2016515508A (ja) 2016-05-30

Similar Documents

Publication Publication Date Title
US20230303985A1 (en) Fusion proteins and methods thereof
Frattini et al. The integrated landscape of driver genomic alterations in glioblastoma
US20220265696A1 (en) Micro-rnas and compositions comprising same for the treatment and diagnosis of serotonin-, adrenalin-, noradrenalin-, glutamate-, and corticotropin-releasing hormone- associated medical conditions
EP2877854B2 (en) Fgfr-tacc fusion proteins and methods thereof
CN103866008B (zh) 编码ncrna的超保守区域
WO2008073290A1 (en) Tp53 gene expression and uses thereof
US20210284996A1 (en) Methods and kit for characterizing the modified base status of a transcriptome
US11332789B2 (en) Compositions and methods for diagnosing and treating hyperlipidemia-related diseases
TWI816712B (zh) 癌促進因子表現抑制劑的有效成分之篩選用試藥及其篩選方法、癌之預防或治療劑的有效成分之篩選用試藥及其篩選方法、癌促進因子表現抑制劑及癌之預防或治療劑
US9127078B2 (en) Methods and compositions using splicing regulatory proteins involved in tumor suppression
US20040005615A1 (en) Amplification and overexpression of oncogenes
CN102317469A (zh) 黑素瘤中的gnaq突变
US20130011411A1 (en) Methods and compositions for the diagnosis, prognosis, and treatment of cancer
EP3778644A2 (en) Fgfr-tacc fusion proteins and methods thereof
WO2015048367A1 (en) Fusion proteins and methods of use thereof
WO2009116860A1 (en) Markers providing prognosis of metastasis among cancer patients
WO2019031637A1 (ko) p53-비돌연변이 암에 대한 암 마커 유전자 및 치료제 스크리닝 방법
US20170298360A1 (en) Lats and breast cancer
JP2009529869A (ja) 癌の検出および処置
Shayler et al. Identification and characterization of early human photoreceptor states and cell-state-specific retinoblastoma-related features
Maliszewska Identification of signaling pathways and specifically miRNAs altered in familial and sporadic forms of medullary thyroid tumors
LaForce Understanding the Role of CLP1 in Messenger RNA Transcription and Neurodegeneration
WO2024062445A1 (en) Treatment and method for predicting disease incidence, progression, and prognosis of glioma

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 14769771

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2014236947

Country of ref document: AU

Date of ref document: 20140313

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2016502111

Country of ref document: JP

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2907152

Country of ref document: CA

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2014769771

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 20157029166

Country of ref document: KR

Kind code of ref document: A