# US 2014/0155399 A1 · BRAF Mutations Conferring Resistance to BRAF Inhibitors > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0155399 A1 - Document ID: US20140155399A1 - Status: Published application - Kind: A1 - Application number: 14/096,954 - Filed: 2013-12-04 - Published: 2014-06-05 - Earliest claimed: 2010-02-25 - Assignees: Dana-Farber Cancer Institute, Inc. - Applicants: Dana-Farber Cancer Institute, Inc. - Inventors: Levi Garraway; Caroline Emery - Web page: https://patentgrep.com/patent/US20140155399A1 ## CPC - C12Q 1/485: {involving kinase} - A61K 31/4184: condensed with carbocyclic rings, e.g. benzimidazoles - A61K 31/437: the heterocyclic ring system containing a five-membered ring having nitrogen as a ring hetero atom, e.g. indolizine, beta-carboline - A61K 31/4439: containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole (nicotine A61K31/465) - A61K 31/5377: not condensed and containing further heterocyclic rings, e.g. timolol - A61P 13/00: Drugs for disorders of the urinary system - A61P 15/00: Drugs for genital or sexual disorders (for disorders of sex hormones A61P5/24); Contraceptives - A61P 17/00: Drugs for dermatological disorders - A61P 19/00: Drugs for skeletal disorders - A61P 21/00: Drugs for disorders of the muscular or neuromuscular system - A61P 25/00: Drugs for disorders of the nervous system - A61P 35/00: Antineoplastic agents - A61P 35/02: specific for leukemia - A61P 35/04: specific for metastasis - A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00 - C12N 9/12: transferring phosphorus containing groups, e.g. kinases (2.7) - C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575) - G01N 33/5011: {for testing antineoplastic activity} - G01N 33/573: for enzymes or isoenzymes - C12Q 2600/106: Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism - C12Q 2600/136: Screening for pharmacological compounds - G01N 2333/912: transferring phosphorus containing groups, e.g. kinases (2.7) - G01N 2500/02: Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The present invention relates to methods, compositions and kits concerning resistance to treatment with an anti-cancer agent, specifically an inhibitor of BRAF. In particular embodiments, the invention concerns mutations in a BRAF sequence that confer resistance to a BRAF inhibitor. Identification of such mutations in a BRAF sequence allows the identification and design of second-generation BRAF inhibitors. Methods and kits for detecting the presence of a mutant BRAF sequence in a sample are also provided. ## Claims ### Claim 1 An isolated mutant BRAF polypeptide having a BRAF activity, wherein said mutant BRAF polypeptide comprises an amino acid sequence having at least one amino acid substitution as compared to a wild type BRAF polypeptide (SEQ ID NO: 2) or a BRAF V600E polypeptide (SEQ ID NO:4), the at least one amino acid substitution conferring resistance to one or more BRAF inhibitors on the mutant BRAF polypeptide. ### Claim 2 (depends on 1) The mutant BRAF polypeptide of claim 1, wherein the at least one amino acid substitution occurs at one or more amino acid positions selected from the group consisting of A29, H72, SI 13, S124, P162, C194, L227, P231, C251, V291, Q329, V483, L485, T521, V528, D587, P655, S657, S683, P686, C696, L697, P722, F738, and C748. ### Claim 3 (depends on 1) The mutant BRAF polypeptide of claim 1, wherein the at least one amino acid substitution is selected from the group consisting of A29V, H72N, SI 131, S124F, P162H, C194*, L227F, P231T, C251F, V291F, Q329K, V483E, L485F, T521K, V528F, D587E, P655T, S657*, S683R, P686Q, P686T, C696*, L697I, P722T, F738L, and C748F. ### Claim 4 (depends on 1) The mutant BRAF polypeptide of claim 1, which has one to five amino acid substitutions as compared to the wild type BRAF polypeptide or the BRAF V600E polypeptide. ### Claim 5 (depends on 1) The mutant BRAF polypeptide of claim 1, wherein the BRAF inhibitor is RAF-265 or PLX4720. ### Claim 6 (depends on 1) The mutant BRAF polypeptide of claim 1, wherein the mutant BRAF polypeptide comprises a substitution at one or more of amino acid positions T521K, V528V or P686Q. ### Claim 7 (depends on 1) A method of identifying a compound that is useful in treating cancer, comprising: (a) providing an assay composition comprising a mutant BRAF polypeptide of claim 1 and a BRAF substrate; (b) contacting the assay composition with a test compound under conditions that permit phosphorylation of the BRAF substrate in the absence of the test compound; and (c) determining the effect of the compound on phosphorylation of the BRAF substrate; wherein downmodulation of phosphorylation of the BRAF substrate as compared to a suitable control identifies the compound as a compound that is useful in treating cancer. ### Claim 8 (depends on 7) The method of claim 7, wherein the BRAF substrate is MEK1 or MEK2. ### Claim 9 (depends on 8) The method of claim 8, wherein phosphorylation of MEK1 or MEK2 is determined using a phospho-specific MEK antibody or by measuring phosphorylation of myelin basic protein (MBP). ### Claim 10 (depends on 1) A method of identifying a compound that is useful in treating cancer, comprising: (a) providing an assay composition comprising a mutant BRAF polypeptide of claim 1 and a BRAF substrate; (b) contacting the assay composition with a test compound under conditions that permit phosphorylation of the BRAF substrate in the absence of the test compound; and (c) determining the effect of the compound on phosphorylation of the BRAF substrate; wherein downmodulation of phosphorylation of the BRAF substrate as compared to a suitable control identifies the compound as a compound that is useful in treating cancer. ### Claim 11 (depends on 10) The method of claim 10, wherein the BRAF substrate is MEK1 or MEK2. ### Claim 12 (depends on 11) The method of claim 11, wherein phosphorylation of MEK1 or MEK2 is determined using a phospho-specific MEK antibody or by measuring phosphorylation of myelin basic protein (MBP). ### Claim 13 (depends on 1) A method of identifying a compound that is useful in treating cancer, comprising: a) providing a cell comprising a mutant BRAF polypeptide of claim 1; b) contacting the cell with a test compound; and c) determining the effect of the compound on phosphorylation of a BRAF substrate or determining the effect of the compound on cell proliferation; wherein downmodulation of phosphorylation of the BRAF substrate or reduction in cell proliferation as compared to a suitable control identifies the compound as a compound that is useful in treating cancer. ### Claim 14 (depends on 1) An expression vector comprising an isolated nucleic acid molecule encoding the mutant BRAF polypeptide of claim 1 having BRAF activity. ### Claim 15 (depends on 14) A host cell comprising the expression vector of claim 14. ### Claim 16 (depends on 15) A cell-based screening method for identifying a test compound as a BRAF inhibitor that inhibits a mutant BRAF polypeptide having BRAF activity, the method comprising contacting the host cell of claim 15 with a test compound, wherein sensitivity of the host cell to the test compound identifies the compound as a BRAF inhibitor that inhibits a mutant BRAF polypeptide having BRAF activity. ### Claim 17 (depends on 16) The method of claim 16, wherein sensitivity of the host cell to the test compound is measured using an assay selected from the group consisting of a cell proliferation assay, a cell viability assay, and a MEK phosphorylation assay, wherein a reduction in cell proliferation, cell viability, or MEK phosphorylation in the presence of the test compound identifies the compound as a BRAF inhibitor that inhibits a mutant BRAF polypeptide having BRAF activity. ### Claim 18 A method of identifying a BRAF inhibitor that inhibits a mutant BRAF polypeptide having BRAF activity, comprising: a) selecting a potential drug using computer-assisted modeling with a three-dimensional crystal or solution structure of a mutant BRAF polypeptide, wherein said mutant BRAF polypeptide comprises an amino acid sequence having at least one amino acid substitution as compared to a wild type BRAF polypeptide or a BRAF V600E polypeptide, the at least one amino acid substitution conferring resistance to one or more BRAF inhibitors on the mutant BRAF polypeptide; b) contacting said potential drug with the mutant BRAF polypeptide; and c) detecting the interaction of said potential drug with the mutant BRAF polypeptide; wherein a compound that is capable of interacting with the mutant BRAF polypeptide is identified as a BRAF inhibitor that inhibits a mutant BRAF polypeptide having BRAF activity. ### Claim 19 (depends on 18) The method of claim 18 wherein the test compound is a member of a library of compounds. ### Claim 20 (depends on 18) The method of claim 18, wherein the mutant BRAF polypeptide is a BRAF polypeptide comprising a substitution at one or more of amino acid positions T521K, V528F, or P686Q. ### Claim 21 A method of treating a subject having cancer, comprising: (a) extracting nucleic acid from cells of the cancer; (b) performing a nucleic acid-based assay to analyze a nucleic acid molecule encoding a BRAF poly peptide; and (c) administering a MEK inhibitor or a BRAF inhibitor that inhibits a mutant BRAF polypeptide having BRAF activity to the subject when the nucleic acid molecule contains nucleotides that alter the identity of an amino acid residue at one or more amino acids of the encoded BRAF polypeptide relative to the amino acid at one or more positions selected from the group consisting of A29, H72, S113, S124, P162, C194, L227, P231, C251, V291, Q329, V483, L485, T521, V528, D587, P655, S657, S683, P686, C696, L697, P722, F738, and C748 of SEQ ID NO:2 or SEQ ID NO:4. ### Claim 22 (depends on 21) The method of claim 21, wherein the MEK inhibitor is selected from the group consisting of CI-1040, AZD6244, PD318088, PD98059, PD334581, RDEA 119, 6-Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinoline-3-carbonitrile and 4-[3-Chloro-4-(1-methyl-1H-imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy-7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile. ### Claim 23 (depends on 21) The method of claim 21, wherein the RAF inhibitor is RAF265 or PLX 4720. ### Claim 24 (depends on 21) The method of claim 21, wherein the cancer is selected from the group consisting of melanoma, leukemias, lymphomas, myelomas, carcinomas, metastatic carcinomas, sarcomas, adenomas, nervous system cancers and genitourinary cancers.