Tyrosine kinome

Abstract

Protein kinases are important signaling molecules involved in tumorigenesis. Mutational analysis of the human tyrosine kinase gene family (98 genes) identified somatic alterations in -20% of colorectal cancers, with the majority of mutations occurring in NTRK3, FES, GUCY2F and a previously uncharacterized tyrosine kinase gene called MCCK/MLK4. Most alterations were in conserved residues affecting key regions of the kinase domain. These data represent a paradigm for the unbiased analysis of signal transducing genes in cancer and provide useful targets for therapeutic intervention.

1 . A method for detecting mutations involved in cancer, comprising: 
 identifying members of a family of genes in a database of human nucleotide sequences based on homology to a known member of the family;    determining nucleotide sequence differences in a selected region of each of the members of the family of genes in matched pairs of an individual's cancer cells and normal cells, said differences identifying members of heightened interest;    determining additional nucleotide sequence differences in the members of heightened interest, either in one or more additional regions outside of the selected region, or in matched pairs of cancer cells and normal cells of additional individuals, or in both.    
     
     
         2 . The method of  claim 1  wherein the selected region encodes a catalytic domain.  
     
     
         3 . The method of  claim 1  wherein the selected region encodes a regulatory domain.  
     
     
         4 . The method of  claim 1  further comprising determining whether a nucleotide sequence difference is synonymous or non-synonymous, wherein a non-synonymous mutation is more likely to be functionally relevant to cancer.  
     
     
         5 . The method of  claim 4  further comprising determining whether a nucleotide difference affects an evolutionarily conserved amino acid residue, wherein such a difference is more likely to be functionally relevant to cancer.  
     
     
         6 . The method of  claim 4  further comprising determining whether a nucleotide difference affects a residue within a catalytic domain, wherein such a difference is more likely to be functionally relevant to cancer.  
     
     
         7 . The method of  claim 4  further comprising determining whether a nucleotide difference affects a first amino acid residue which is a positional equivalent of a second amino acid residue in a protein encoded by another member of the family of genes, wherein mutation of said second residue in the protein encoded by another member of the family causes disease, wherein such a difference affecting the first amino acid is more likely to be functionally relevant to cancer.  
     
     
         8 . The method of  claim 4  further comprising determining whether a nucleotide difference affects a first amino acid residue which is within 5 amino acid residues of an equivalent of a second amino acid residue in a protein encoded by another member of the family of genes, wherein mutation of said second residue in the protein encoded by another member of the family causes disease, wherein such a difference affecting the first amino acid is more likely to be functionally relevant to cancer.  
     
     
         9 . The method of  claim 8  wherein the first amino acid residue is within 3 amino acid residues of the equivalent of the second amino acid residue.  
     
     
         10 . The method of  claim 8  wherein the first amino acid residue is within 1 amino acid residues of the equivalent of the second amino acid residue.  
     
     
         11 . A method of screening test substances for use as anti-cancer agents, comprising: 
 contacting a test substance with an activated protein kinase selected from the group consisting of: NTRK3, FES, MCCK, EPHA3, NTRK2, INSRR, JAK1, PDGFRA, EPHA7, EPHA8, KDR, FGFR1, and ERBB4;    testing activity of the activated protein kinase, wherein a test substance which inhibits the activity of the activated protein kinase is a potential anti-cancer agent.    
     
     
         12 . The method of  claim 11  wherein the activated protein kinase is in a cell.  
     
     
         13 . The method of  claim 11  wherein the activated protein kinase is isolated from a cell.  
     
     
         14 . The method of  claim 11  wherein the activated protein kinase is in a cell of a cancer cell line.  
     
     
         15 . The method of  claim 11  wherein the activated protein kinase is in a cell which has been modified to express the activated protein kinase.  
     
     
         16 . The method of  claim 11  wherein the activated protein kinase is NTRK3.  
     
     
         17 . The method of  claim 11  wherein the activated protein kinase is FES.  
     
     
         18 . The method of  claim 11  wherein the activated protein kinase is MCCK.  
     
     
         19 . The method of  claim 11  wherein the activated protein kinase is EPHA3.  
     
     
         20 . The method of  claim 11  wherein the activated protein kinase is NTRK2.  
     
     
         21 . The method of  claim 11  wherein the activated protein kinase is INSRR.  
     
     
         22 . The method of  claim 11  wherein the activated protein kinase is JAK1.  
     
     
         23 . The method of  claim 11  wherein the activated protein kinase is PDGFRA.  
     
     
         24 . The method of  claim 11  wherein the activated protein kinase is EPHA7.  
     
     
         25 . The method of  claim 11  wherein the activated protein kinase is EPHA8.  
     
     
         26 . The method of  claim 11  wherein the activated protein kinase is ERBB4.  
     
     
         27 . The method of  claim 11  wherein the activated protein kinase is FGFR1.  
     
     
         28 . The method of  claim 11  wherein the activated protein kinase is KDR.  
     
     
         29 . The method of  claim 16  wherein the activated NTRK3 protein kinase has a mutation from the group consisting of I695V, G608S, L760I, K732T, and R731Q.  
     
     
         30 . The method of  claim 17  wherein the activated FES protein kinase has a mutation from the group consisting of M704V, R706Q, V743M, and S759F.  
     
     
         31 . The method of  claim 18  wherein the activated MCCK protein kinase has a mutation from the group consisting of H261Y, H261Q, G291E, A293E, W296STP, R470C, R553Stp, N596I, and K629E.  
     
     
         32 . The method of  claim 19  wherein the activated EPHA3 protein kinase has a mutation from the group consisting of S792P and D806N.  
     
     
         33 . The method of  claim 20  wherein the activated NTRK2 protein kinase has a mutation from the group consisting of T695I and D751N.  
     
     
         34 . The method of  claim 21  wherein the activated INSRR protein kinase has a mutation T985M.  
     
     
         35 . The method of  claim 22  wherein the activated JAK1 protein kinase has a mutation E886K.  
     
     
         36 . The method of  claim 23  wherein the activated PGDFRA protein kinase is the result of a G→A splice site mutation at position 1 of the donor site of exon 15.  
     
     
         37 . The method of  claim 24  wherein the activated EPHA7 protein kinase has a mutation S768I.  
     
     
         38 . The method of  claim 25  wherein the activated EPHA8 protein kinase has a mutation D873N.  
     
     
         39 . The method of  claim 26  wherein the activated ERBB4 protein kinase is I1030M.  
     
     
         40 . The method of  claim 27  wherein the activated FGFR1 protein kinase has a mutation A429S.  
     
     
         41 . The method of  claim 28  wherein the activated KDR protein kinase has a mutation selected from the group consisting of G800D, R819Stop, and A1073T.  
     
     
         42 . A method of screening test substances for use as anti-cancer agents, comprising: 
 contacting a test substance with a mutated GUCY2F guanylate cyclase;    testing activity of the mutated GUCY2F guanylate cyclase, wherein a test substance which increases the activity of the mutated GUCY2F guanylate cyclase is a potential anti-cancer agent.    
     
     
         43 . The method of  claim 42  wherein the mutated GUCY2F guanylate cyclase is in a cell.  
     
     
         44 . The method of  claim 42  wherein the mutated GUCY2F guanylate cyclase is isolated from a cell.  
     
     
         45 . The method of  claim 42  wherein the mutated GUCY2F guanylate cyclase is in a cell of a cancer cell line.  
     
     
         46 . The method of  claim 42  wherein the mutated GUCY2F guanylate cyclase is in a cell which has been modified to express the activated protein kinase.  
     
     
         47 . The method of  claim 42  wherein the mutated GUCY2F guanylate cyclase has a mutation selected from the group consisting of D225Y, A360T, Q361H, F390L, R492H, R545S, E624D, E778G, the result of a T→C splice site mutation at position 2 of the donor splice site of exon 17, and V1026M.  
     
     
         48 . An isolated, activated protein kinase selected from the group consisting of: 
 NTRK3, FES, MCCK, GUCY2F, EPHA3, NTRK2, INSRR, JAK1, PDGFRA, EPHA7, EPHA8, KDR, FGFR1, and ERBB4.    
     
     
         49 . The isolated, activated tyrosine of  claim 48  wherein the activated protein kinase is NTRK3.  
     
     
         50 . The isolated, activated tyrosine of  claim 48  wherein the activated protein kinase is FES.  
     
     
         51 . The isolated, activated tyrosine of  claim 48  wherein the activated protein kinase is MCCK.  
     
     
         52 . The isolated, activated tyrosine of  claim 48  wherein the activated protein kinase is EPHA3.  
     
     
         53 . The isolated, activated tyrosine of  claim 48  wherein the activated protein kinase is NTRK2.  
     
     
         54 . The isolated, activated tyrosine of  claim 48  wherein the activated protein kinase is INSRR.  
     
     
         55 . The isolated, activated tyrosine of  claim 48  wherein the activated protein kinase is JAK1.  
     
     
         56 . The isolated, activated tyrosine of  claim 48  wherein the activated protein kinase is PDGFRA.  
     
     
         57 . The isolated, activated tyrosine of  claim 48  wherein the activated protein kinase is EPHA7.  
     
     
         58 . The isolated, activated tyrosine of  claim 48  wherein the activated protein kinase is EPHA8.  
     
     
         59 . The isolated, activated tyrosine of  claim 48  wherein the activated protein kinase is ERBB4.  
     
     
         60 . The isolated, activated tyrosine of  claim 48  wherein the activated protein kinase is KDR.  
     
     
         61 . The isolated, activated tyrosine of  claim 48  wherein the activated protein kinase is FGFR1.  
     
     
         62 . The isolated, activated tyrosine of  claim 49  wherein the activated NTRK3 protein kinase has a mutation from the group consisting of I695V, G608S, L760I, K732T, and R731Q.  
     
     
         63 . The isolated, activated tyrosine of  claim 50  wherein the activated FES protein kinase has a mutation from the group consisting of M704V, R706Q, V743M, and S759F.  
     
     
         64 . The isolated, activated tyrosine of  claim 51  wherein the activated MCCK protein kinase has a mutation from the group consisting of H261Y, H261Q, G291E, A293E, W296STP, R470C, R470C, R553Stp, N596I, and K629E.  
     
     
         65 . The isolated, activated tyrosine of  claim 52  wherein the activated EPHA3 protein kinase has a mutation from the group consisting of S792P and D806N.  
     
     
         66 . The isolated, activated tyrosine of  claim 53  wherein the activated NTRK2 protein kinase has a mutation from the group consisting of T695I and D751N.  
     
     
         67 . The isolated, activated tyrosine of  claim 54  wherein the activated INSRR protein kinase has a mutation T985M.  
     
     
         68 . The isolated, activated tyrosine of  claim 55  wherein the activated JAK1 protein kinase has a mutation E886K.  
     
     
         69 . The isolated, activated tyrosine of  claim 56  wherein the activated PGDFRA protein kinase is the result of a G→A splice site mutation at position 1 of the donor site of exon 15.  
     
     
         70 . The isolated, activated tyrosine of  claim 57  wherein the activated EPHA7 protein kinase has a mutation S768I.  
     
     
         71 . The isolated, activated tyrosine of  claim 58  wherein the activated EPHA8 protein kinase has a mutation D873N.  
     
     
         72 . The isolated, activated tyrosine of  claim 59  wherein the activated ERBB4 protein kinase has a mutation I1030M.  
     
     
         73 . The isolated, activated tyrosine of  claim 60  wherein the activated KDR protein kinase has a mutation selected from the group consisting of G800D, R819Stop, and A1073T.  
     
     
         74 . The isolated, activated tyrosine of  claim 61  wherein the activated FGFR1 protein kinase has a mutation A429S.  
     
     
         75 . An isolated, mutated GUCY2F protein.  
     
     
         76 . The isolated, mutated GUCY2F protein of  claim 75  which has a mutation from the group consisting of D225Y, A360T, Q361H, F390L, R492H, R545S, E624D, E778G, the result of a T→C splice site mutation at position 2 of the donor splice site of exon 17, and V1026M.  
     
     
         77 . A method of categorizing cancers, comprising: 
 determining the sequence of one or more protein kinase family members selected from the group consisting of NTRK3, FES, MCCK, EPHA3, NTRK2, INSRR, JAK1, PDGFRA, EPHA7, EPHA8, GUCY2F, KDR, FGFR1, and ERBB4 in a sample of a cancer tissue;    identifying a somatic mutation of said one or more protein kinase family members in the cancer tissue;    assigning the cancer tissue to a set based on the presence of the somatic mutation.    
     
     
         78 . The method of  claim 77  wherein the mutation is one which activates protein kinase activity.  
     
     
         79 . The method of  claim 78  wherein the protein kinase family member is NTRK3.  
     
     
         80 . The method of  claim 78  wherein the protein kinase family member is FES.  
     
     
         81 . The method of  claim 78  wherein the protein kinase family member is MCCK.  
     
     
         82 . The method of  claim 78  wherein the protein kinase family member is EPHA3.  
     
     
         83 . The method of  claim 78  wherein the protein kinase family member is NTRK2.  
     
     
         84 . The method of  claim 78  wherein the protein kinase family member is INSSR.  
     
     
         85 . The method of  claim 78  wherein the protein kinase family member is JAK1.  
     
     
         86 . The method of  claim 78  wherein the protein kinase family member is PDGFRA.  
     
     
         87 . The method of  claim 78  wherein the protein kinase family member is EPHA7.  
     
     
         88 . The method of  claim 77  wherein the protein kinase family member is GUCY2F.  
     
     
         89 . The method of  claim 78  wherein the protein kinase family member is ERBB4.  
     
     
         90 . The method of  claim 78  wherein the protein kinase family member is EPHA8.  
     
     
         91 . The method of  claim 78  wherein the protein kinase family member is KDR.  
     
     
         92 . The method of  claim 78  wherein the protein kinase family member is FGFR1.  
     
     
         93 . The method of  claim 77  wherein the set is used to analyze or design clinical trials.  
     
     
         94 . The method of  claim 77  wherein the set is used to correlate with prognostic data.  
     
     
         95 . The method of  claim 77  wherein the set is used to correlate with recurrence data.  
     
     
         96 . The method of  claim 77  wherein the set is used to select an appropriate therapeutic agent.