NOTCH mutations leading to increased receptor signaling

Abstract

The present invention is based upon the identification of regions within the NOTCH-1 receptor that, when mutated, lead to increased receptor signaling. The mutations are associated with uncontrolled cellular growth and this growth may be arrested using agents that interfere with NOTCH-1 activity, such as inhibitors of gamma-secretase. Assays for the NOTCH-1 mutations may be used diagnostically or as part of a treatment regimen for cancer patients.

1 . A substantially purified mutant NOTCH-1 receptor, comprising the amino acid sequence of SEQ ID NO:1 except that said amino acid sequence has one or more mutations in amino acids 1571-1622 and/or 1674-1730 and/or amino acid 2311 to amino acid 2556 such that, relative to unmutated NOTCH-1, said mutant NOTCH-1 receptor exhibits increased signaling in response to incubation with gamma-secretase.  
     
     
         2 . The substantially purified mutant receptor of  claim 1 , wherein, except for said one or more mutations, said mutant receptor consists of the amino sequence of SEQ ID NO:1.  
     
     
         3 . The substantially purified mutant NOTCH-1 receptor of  claim 1 , wherein said one or more mutations include one or more of the following: 
 a) the portion of the amino acid sequence of SEQ ID NO:1 from amino acid 1571 to amino acid 1618 is replaced with a sequence selected from the group consisting of SEQ ID NO:12-SEQ ID NO:22, SEQ ID NO:102-118, and SEQ ID NO:145;    b) the portion of the amino acid sequence of SEQ ID NO:1 from amino acid 1571 to amino acid 1622 is replaced with a sequence selected from the group consisting of SEQ ID NO:23- SEQ ID NO:29, or SEQ ID NO:119-127;    c) the portion of the amino acid sequence of SEQ ID NO:1 from amino acid 1674 to amino acid 1730 is replaced with a sequence selected from the group consisting of SEQ ID NO:70- SEQ ID NO:75, or SEQ ID NO:128-137;    d) the portion of the amino acid sequence of SEQ ID NO:1 from amino acid 2311 to amino acid 2556 is replaced with a sequence selected from the group consisting of SEQ ID NO:30- SEQ ID NO:59 or SEQ ID NO:138-144.    
     
     
         4 . The substantially purified mutant receptor of  claim 2 , wherein, except for said one or more mutations, said mutant receptor consists of the amino sequence of SEQ ID NO:1.  
     
     
         5 . The substantially purified mutant NOTCH-1 receptor of  claim 1 , wherein said sequence is mutated at one or more amino acid positions selected from the group consisting of: 1571; 1573; 1575; 1576; 1578; 1579; 1583; 1586; 1588; 1590; 1593; 1594; 1595; 1598; 1600-1607; 1610-1618; 1674; 1675; 1677; 1679-1684; 1686; 1695; 1696; 1703; 1706; 1715; 1720; 1725; and 1728.  
     
     
         6 . The substantially purified receptor of  claim 5 , wherein, except for said one or more mutated amino acids, said receptor consists of the amino acid sequence of SEQ ID NO:1.  
     
     
         7 . A substantially purified polynucleotide comprising a nucleotide sequence encoding the mutant NOTCH-1 receptor of  claim 1 .  
     
     
         8 . The substantially purified polynucleotide of  claim 7 , wherein said polynucleotide consists of a nucleotide sequence encoding the mutant receptor of  claim 1 .  
     
     
         9 . A substantially purified polynucleotide, at least 15 nucleotides in length which hybridizes to the polynucleotide of  claim 8  under stringent conditions but which does not hybridize to a polynucleotide encoding unmutated NOTCH-1 under the same conditions.  
     
     
         10 . A vector comprising a promoter operably linked to a polynucleotide encoding the mutant NOTCH-1 receptor of  claim 1 .  
     
     
         11 . A host cell transformed with the vector of  claim 10 .  
     
     
         12 . An antibody that binds to the mutant NOTCH-1 receptor of  claim 1  with at least a hundredfold greater affinity than to the unmutated NOTCH-1 receptor of SEQ ID NO:1.  
     
     
         13 . A method of diagnostically testing a biological sample for the presence of cancer cells, comprising assaying said biological sample for the mutant NOTCH-1 receptor of  claim 1 .  
     
     
         14 . A method of diagnostically testing a biological sample for the presence of cancer cells, comprising assaying said biological sample for cells expressing the polynucleotide of  claim 7 .  
     
     
         15 . A method of diagnostically testing a biological sample for the presence of cancer cells, comprising assaying said biological sample for cells expressing the polynucleotide of  claim 8 .  
     
     
         16 . The method of  claim 15 , wherein said method is performed by PCR amplifying either the full length polynucleotide encoding said mutant NOTCH-1 receptor or a portion of the polynucleotide encoding said mutant NOTCH-1 receptor.  
     
     
         17 . A method of slowing or stopping the growth of cells identified as having the mutant NOTCH-1 receptor of  claim 1 , comprising contacting said cells with an effective amount of a gamma-secretase inhibitor.  
     
     
         18 . A method of treating a patient for cancer comprising: 
 a) assaying a biological sample from said patient for the presence of cancer cells having the mutant Notch-1 receptor of  claim 1;     b) treating said patient with an effective amount of an agent that inhibits Notch-1 receptor signaling if said biological sample is found to have said mutant Notch-1 receptor.    
     
     
         19 . The method of  claim 18 , wherein said cancer is T-cell acute lymphoblastic leukemia.  
     
     
         20 . The method of  claim 18 , wherein said agent is a gamma-secretase inhibitor.  
     
     
         21 . A method of assaying a test compound for its ability to inhibit abnormal cellular growth induced by the presence of a mutated NOTCH-1 receptor, comprising: 
 a) incubating said test compound with cells that express the mutant NOTCH-1 receptor of  claim 1 , said incubation being carried out in the presence of gamma-secretase;    b) comparing the amount of receptor activity that occurs as the result of the incubation of step a) with the amount of activity that occurs in a similar incubation carried out in the absence of said test compound; and    c) concluding that said test compound should inhibit the growth of cells expressing mutant NOTCH-1 receptor if the amount of activity observed in the presence of said test compound is less than the amount of activity observed in the absence of said test compound.