Inhibitors of human ezh2, and methods of use thereof

Abstract

The invention relates to inhibition of wild-type and certain mutant forms of human histone methyltransferase EZH2, the catalytic subunit of the PRC2 complex which catalyzes the mono- through tri-methylation of lysine 27 on histone H3 (H3-K27). In one embodiment the inhibition is selective for the mutant form of the EZH2, such that trimethylation of H3-K27, which is associated with certain cancers, is inhibited. The methods can be used to treat cancers including follicular lymphoma and diffuse large B-cell lymphoma (DLBCL). Also provided are methods for identifying small molecule selective inhibitors of the mutant forms of EZH2 and also methods for determining responsiveness to an EZH2 inhibitor in a subject.

1 . A method comprising
 detecting the presence of a mutation in the EZH2 substrate pocket domain as defined in SEQ ID NO: 7 in a sample from a subject having a cancer or a precancerous condition by contacting the sample with at least one primer that specifically hybridizes to a portion of the nucleic acid encoding SEQ ID NO: 7, wherein the mutation increases EZH2 trimethylation of Lys27 of histone H3 (H3-K27);   identifying the subject as a candidate for treatment with an EZH2 inhibitor based on the presence of said EZH2 mutation; and   administering a therapeutically effective amount of an EZH2 inhibitor to the identified subject.   
     
     
         2 . (canceled) 
     
     
         3 . A method comprising
 detecting the presence of a mutation in the EZH2 substrate pocket domain as defined in SEQ ID NO: 7 in a sample from a subject having a cancer or a precancerous condition by contacting the sample with at least one primer that specifically hybridizes to a portion of the nucleic acid encoding SEQ ID NO: 7, wherein the mutation increases EZH2 trimethylation of Lys27 of histone H3 (H3-K27);   identifying the subject as a candidate for treatment with an EZH2 inhibitor based on the presence of said EZH2 mutation, and   selecting a therapy that includes the administration of a therapeutically effective amount of an EZH2 inhibitor to the identified subject.   
     
     
         4 . A method comprising
 detecting the presence of a mutation in the EZH2 substrate pocket domain as defined in SEQ ID NO: 7, in a sample from a subject having a cancer or a precancerous condition, wherein the mutation increases EZH2 trimethylation of Lys27 of histone H3 (H3-K27);   identifying the subject as a candidate for treatment with an EZH2 inhibitor based on the presence of said EZH2 mutation;   administering a therapeutically effective amount of an EZH2 inhibitor to the identified subject.   
     
     
         5 . The method of  claim 1 , wherein the nucleic acid encoding SEQ ID NO: 7 is SEQ ID NO: 8. 
     
     
         6 . (canceled) 
     
     
         7 . The method of  claim 3 , wherein the nucleic acid encoding SEQ ID NO: 7 is SEQ ID NO: 8. 
     
     
         8 . The method of  claim 4 , wherein the nucleic acid encoding SEQ ID NO: 7 is SEQ ID NO: 8. 
     
     
         9 . The method of  claim 1 , wherein the mutation is a substitution mutation at amino acid position 677, 687, 674, 685, or 641 of SEQ ID NO: 7. 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 3 , wherein the mutation is a substitution mutation at amino acid position 677, 687, 674, 685, or 641 of SEQ ID NO: 7. 
     
     
         12 . The method of  claim 4 , wherein the mutation is a substitution mutation at amino acid position 677, 687, 674, 685, or 641 of SEQ ID NO: 7. 
     
     
         13 . The method of  claim 9 , wherein said mutation is selected from the group consisting of a substitution of glycine (G) for the wild type residue alanine (A) at amino acid position 677 of SEQ ID NO: 7 (A677G); a substitution of valine (V) for the wild type residue alanine (A) at amino acid position 687 of SEQ ID NO: 7 (A687V); a substitution of methionine (M) for the wild type residue valine (V) at amino acid position 674 of SEQ ID NO: 7 (V674M); a substitution of histidine (H) for the wild type residue arginine (R) at amino acid position 685 of SEQ ID NO: 7 (R685H); a substitution of cysteine (C) for the wild type residue arginine (R) at amino acid position 685 of SEQ ID NO: 7 (R685C); a substitution of phenylalanine (F) for the wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 7 (Y641F); a substitution of histidine (H) for the wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 7 (Y641H); a substitution of asparagine (N) for the wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 7 (Y641N); a substitution of serine (S) for the wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 7 (Y641S); and a substitution of cysteine (C) for the wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 7 (Y641C). 
     
     
         14 . (canceled) 
     
     
         15 . The method of  claim 11 , wherein said mutation is selected from the group consisting of a substitution of glycine (G) for the wild type residue alanine (A) at amino acid position 677 of SEQ ID NO: 7 (A677G); a substitution of valine (V) for the wild type residue alanine (A) at amino acid position 687 of SEQ ID NO: 7 (A687V); a substitution of methionine (M) for the wild type residue valine (V) at amino acid position 674 of SEQ ID NO: 7 (V674M); a substitution of histidine (H) for the wild type residue arginine (R) at amino acid position 685 of SEQ ID NO: 7 (R685H); a substitution of cysteine (C) for the wild type residue arginine (R) at amino acid position 685 of SEQ ID NO: 7 (R685C); a substitution of phenylalanine (F) for the wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 7 (Y641F); a substitution of histidine (H) for the wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 7 (Y641H); a substitution of asparagine (N) for the wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 7 (Y641N); a substitution of serine (S) for the wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 7 (Y641S); and a substitution of cysteine (C) for the wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 7 (Y641C). 
     
     
         16 . The method of  claim 12 , wherein said mutation is selected from the group consisting of a substitution of glycine (G) for the wild type residue alanine (A) at amino acid position 677 of SEQ ID NO: 7 (A677G); a substitution of valine (V) for the wild type residue alanine (A) at amino acid position 687 of SEQ ID NO: 7 (A687V); a substitution of methionine (M) for the wild type residue valine (V) at amino acid position 674 of SEQ ID NO: 7 (V674M); a substitution of histidine (H) for the wild type residue arginine (R) at amino acid position 685 of SEQ ID NO: 7 (R685H); a substitution of cysteine (C) for the wild type residue arginine (R) at amino acid position 685 of SEQ ID NO: 7 (R685C); a substitution of phenylalanine (F) for the wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 7 (Y641F); a substitution of histidine (H) for the wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 7 (Y641H); a substitution of asparagine (N) for the wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 7 (Y641N); a substitution of serine (S) for the wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 7 (Y641S); and a substitution of cysteine (C) for the wild type residue tyrosine (Y) at amino acid position 641 of SEQ ID NO: 7 (Y641C). 
     
     
         17 . The method of  claim 1 , wherein the EZH2 inhibitor is a small molecule. 
     
     
         18 . (canceled) 
     
     
         19 . The method of  claim 3 , wherein the EZH2 inhibitor is a small molecule. 
     
     
         20 . The method of  claim 4 , wherein the EZH2 inhibitor is a small molecule. 
     
     
         21 . The method of  claim 1 , wherein the cancer is lymphoma, selected from the group consisting of non-Hodgkin lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma of germinal center B cell-like subtype. 
     
     
         22 . (canceled) 
     
     
         23 . The method of  claim 3 , wherein the cancer is lymphoma, selected from the group consisting of non-Hodgkin lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma of germinal center B cell-like subtype. 
     
     
         24 . The method of  claim 4 , wherein the cancer is lymphoma, selected from the group consisting of non-Hodgkin lymphoma, follicular lymphoma, and diffuse large B-cell lymphoma of germinal center B cell-like subtype. 
     
     
         25 . The method of  claim 1  wherein the EZH2 inhibitor is 
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt thereof. 
     
     
         26 . (canceled) 
     
     
         27 . The method of  claim 3  wherein the EZH2 inhibitor is 
       
         
           
           
               
               
           
         
       
       or a pharmaceutically acceptable salt thereof. 
     
     
         28 . (canceled)