# US 2009/0269751 A1 · Dot1 histone methyltransferases as a target for identifying therapeutic agents for leukemia > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2009/0269751 A1 - Document ID: US20090269751A1 - Status: Published application - Kind: A1 - Application number: 12/251,037 - Filed: 2008-10-14 - Published: 2009-10-29 - Earliest claimed: 2003-06-13 - Assignees: The University of North Carolina at Chapel Hill - Inventors: YI ZHANG; QIN FENG; YUKI OKADA; GUOLIANG XU - Web page: https://patentgrep.com/patent/US20090269751A1 ## CPC - C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575) - C07H 21/04: with deoxyribosyl as saccharide radical - C12N 9/1007: {Methyltransferases (general) (2.1.1.)} - C12Q 2600/118: Prognosis of disease development - C12Q 2600/136: Screening for pharmacological compounds - C12Q 2600/154: Methylation markers - G01N 33/53: Immunoassay; Biospecific binding assay; Materials therefor - G01N 33/6893: {related to diseases not provided for elsewhere} - G01N 2440/12: alkylation, e.g. methylation, (iso-)prenylation, farnesylation - 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 - C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13) - 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 provides polypeptides with histone H3 lysine 79 methyltransferase activity as well as nucleic acids encoding the same. Also provided are methods of using the polypeptides and nucleic acids of the invention in screening assays to identify compounds of interest. Further provided are diagnostic methods for leukemia and prognostic methods to predict the course of the disease in a subject. ## Claims ### Claim 1 An isolated nucleic acid comprising a nucleotide sequence encoding a DOT1L polypeptide, said nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:1; (b) a nucleotide sequence that hybridizes to the complete complement of a nucleotide sequence comprising the nucleotide sequence of SEQ ID NO:1 under stringent conditions, wherein said nucleotide sequence encodes a polypeptide having histone H3 lysine 79 (H3-K79) methyltransferase activity; (c) a nucleotide sequence having at least 95% nucleotide sequence similarity with the nucleotide sequence of SEQ ID NO:1, wherein said nucleotide sequence encodes a polypeptide having H3-K79 methyltransferase activity; (d) a nucleotide sequence that differs from the nucleotide sequence of (a) above due to the degeneracy of the genetic code; (f) a nucleotide sequence comprising a functional fragment of SEQ ID NO:1, wherein said functional fragment comprises at least the coding region of the histone methyltransferase catalytic domain of SEQ ID NO:1 or a nucleotide sequence that hybridizes to the complete complement of the coding region of the histone methyltransferase catalytic domain of SEQ ID NO:1 under stringent conditions, and wherein said functional fragment encodes a polypeptide having H3-K79 methyltransferase activity; (g) a nucleotide sequence comprising a functional fragment of SEQ ID NO:1, wherein said functional fragment comprises at least the coding region of the histone methyltransferase catalytic domain of SEQ ID NO:1 or a nucleotide sequence having at least 95% nucleotide sequence similarity thereto, and wherein said functional fragment encodes a polypeptide having H3-K79 methyltransferase activity; and (h) a nucleotide sequence comprising a functional fragment of SEQ ID NO:1, wherein said functional fragment comprises at least the coding region of the histone methyltransferase catalytic domain of SEQ ID NO:1 or a nucleotide sequence that differs therefrom due to the degeneracy of the genetic code, and wherein said functional fragment encodes a polypeptide having H3-K79 methyltransferase activity. ### Claim 2 (depends on 1) The isolated nucleic acid of claim 1, wherein the nucleotide sequence encodes the DOT1L polypeptide of SEQ ID NO:2 or a DOT1L polypeptide having at least 95% amino acid sequence similarity with the amino acid sequence of SEQ ID NO:2. ### Claim 3 (depends on 2) The isolated nucleic acid of claim 2, wherein the nucleotide sequence encodes the DOT1L polypeptide of SEQ ID NO:2. ### Claim 4 (depends on 1) The isolated nucleic acid of claim 1, wherein the nucleotide sequence comprises SEQ ID NO:1. ### Claim 5 (depends on 1) The isolated nucleic acid of claim 1, wherein the nucleotide sequence consists of SEQ ID NO:1. ### Claim 6 (depends on 1) The isolated nucleic acid of claim 1, wherein the isolated nucleic acid encodes a DOT1L fusion protein. ### Claim 7 (depends on 1) A vector comprising the isolated nucleic acid of claim 1. ### Claim 8 (depends on 7) The vector of claim 7, wherein the vector is an expression vector. ### Claim 9 (depends on 1) A cell comprising the isolated nucleic acid of claim 1. ### Claim 10 (depends on 8) A cell comprising the vector of claim 8. ### Claim 11 (depends on 10) A method of producing a DOT1L protein, comprising culturing the cell of claim 10 in vitro such that DOT1L protein is expressed from said nucleotide sequence encoding the DOT1L polypeptide. ### Claim 12 A method of identifying a compound that modulates DOT1L histone H3 lysine 79 (H3-K79) methyltransferase activity, comprising: contacting a DOT1L polypeptide with a nucleosome substrate comprising H3 in the presence of a test compound; wherein the DOT1L polypeptide comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:2; (b) an amino acid sequence having at least 95% amino acid sequence similarity with the amino acid sequence of SEQ ID NO:2, wherein said DOT1L polypeptide has H3-K79 methyltransferase activity; and (c) a functional fragment of at least 400 amino acids of the amino acid sequence of (a) or (b) above, wherein said functional fragment comprises a DOT1L histone methyltransferase catalytic domain and has H3-K79 methyltransferase activity; detecting the level of H3-K79 methylation of the nucleosome substrate under conditions sufficient to provide H3-K79 methylation, wherein an elevation or reduction in H3-K79 methylation in the presence of the test compound as compared with the level of histone H3-K79 methylation in the absence of the test compound indicates that the test compound modulates DOT1L H3-K79 methyltransferase activity. ### Claim 13 (depends on 12) The method of claim 12, wherein the test compound enhances DOT1L H3-K79 methyltransferase activity. ### Claim 14 (depends on 12) The method of claim 12, wherein the test compound is an inhibitor of DOT1L H3-K79 methyltransferase activity. ### Claim 15 (depends on 12) The method of claim 12, wherein the method is carried out as a cell-free assay. ### Claim 16 (depends on 12) The method of claim 12, wherein the method is carried out as a cell-based assay. ### Claim 17 (depends on 16) The method of claim 16, wherein the DOT1L polypeptide is expressed from a nucleic acid. ### Claim 18 A method of identifying a compound that inhibits binding of DOT1L polypeptide to a second polypeptide, comprising: contacting a DOT1L polypeptide with a second polypeptide in the presence of a test compound; wherein the DOT1I polypeptide comprises an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of SEQ ID NO:2; (b) an amino acid sequence having at least 95% amino acid sequence similarity with the amino acid sequence of SEQ ID NO:2, wherein said DOT1L polypeptide has H3-K79 methyltransferase activity; and (c) a functional fragment of at least 400 amino acids of the amino acid sequence of (a) or (b) above, wherein said functional fragment comprises a DOT1L histone methyltransferase catalytic domain and has H3-K79 methyltransferase activity; detecting the level of binding between DOT1L polypeptide and the second polypeptide under conditions sufficient for binding of DOT1L polypeptide to the second polypeptide, wherein a reduction in binding between DOT1L polypeptide and the second polypeptide in the presence of the test compound as compared with the level of binding in the absence of the test compound indicates that the test compound inhibits binding of DOT1L polypeptide to the second polypeptide. ### Claim 19 (depends on 18) The method of claim 18, wherein the second polypeptide is AF10 or an MLL fusion protein. ### Claim 20 (depends on 19) The method of claim 19, wherein the second polypeptide is an MLL-AF10 fusion protein.