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 .- 28 . (canceled) 
     
     
         29 . A method, comprising detecting the presence of a Y641F, Y641H, Y641N, or Y641S substitution mutation in an enhancer of Zeste Homolog 2 (EZH2) protein of SEQ ID NO: 1 or at corresponding amino acid positions in isoform a (SEQ ID NO: 3), isoform b (SEQ ID NO: 5) or isoform e (SEQ ID NO: 21), wherein said EZH2 protein is in a biological sample obtained from a subject having lymphoma. 
     
     
         30 . The method of  claim 29 , further comprising the step of detecting the wild-type EZH2 allele. 
     
     
         31 . The method of  claim 29 , wherein the mutation increases EZH2 trimethylation of Lys27 of histone H3 (H3-K27). 
     
     
         32 . The method of  claim 29 , wherein the method further comprises detecting the presence of a substitution mutation at amino acid position 677, 687, 674, or 685 of SEQ ID NO: 1 or at corresponding positions in isoform a (SEQ ID NO: 3), isoform b (SEQ ID NO: 5) or isoform e (SEQ ID NO: 21). 
     
     
         33 . The method of  claim 29 , wherein the substitution mutation is in the EZH2 substrate pocket domain of SEQ ID NO: 6. 
     
     
         34 . The method of  claim 33 , wherein the EZH2 substrate pocket domain comprises the SET domain set forth in SEQ ID NO: 7. 
     
     
         35 . The method of  claim 33 , wherein the SEQ ID NO: 7 is comprised by SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 21. 
     
     
         36 . The method of  claim 32 , wherein the mutant EZH2 comprises a substitution of glycine (G) for the wild type residue alanine (A) at amino acid position 677 (A677G) of SEQ ID NO: 1 or at a corresponding position in isoform a (SEQ ID NO: 3), isoform b (SEQ ID NO: 5) or isoform e (SEQ ID NO: 21). 
     
     
         37 . The method of  claim 32  wherein the mutant EZH2 comprises a substitution of valine (V) for the wild type residue alanine (A) at amino acid position 687 (A687V) of SEQ ID NO: 1 or at a corresponding position in isoform a (SEQ ID NO: 3), isoform b (SEQ ID NO: 5) or isoform e (SEQ ID NO: 21). 
     
     
         38 . The method of  claim 32 , wherein the mutant EZH2 comprises a substitution of methionine (M) for the wild type residue valine (V) at amino acid position 674 (V674M) of SEQ ID NO: 1 or at a corresponding position in isoform a (SEQ ID NO: 3), isoform b (SEQ ID NO: 5) or isoform e (SEQ ID NO: 21). 
     
     
         39 . The method of  claim 32 , wherein the mutant EZH2 comprises a substitution of histidine (H) or cysteine (C) for the wild type residue arginine (R) at amino acid position 685 (R685H or R685C) of SEQ ID NO: 1 or at a corresponding position in isoform a (SEQ ID NO: 3), isoform b (SEQ ID NO: 5) or isoform e (SEQ ID NO: 21). 
     
     
         40 . The method of  claim 29 , wherein the mutant EZH2 comprises a substitution of phenylalanine (F), histidine (H), asparagine (N), serine (S), or cysteine (C) for the wild type residue tyrosine (Y) at amino acid position 641. 
     
     
         41 . The method of  claim 29 , wherein the EZH2 inhibitor is a compound of Table 1, or a pharmaceutically acceptable salt thereof. 
     
     
         42 . The method of  claim 29 , wherein the EZH2 inhibitor is 
       
         
           
           
               
               
           
         
       
     
     
         43 . The method of  claim 29 , wherein the detecting comprises:
 a. contacting the biological sample with at least one antibody that binds specifically to the EZH2 comprising the mutation in the substrate pocket domain, wherein the at least one antibody and the mutant EZH2 form a complex; and   b. detecting the presence of the complex.   
     
     
         44 . The method of  claim 43 , further comprising identifying the subject as a candidate for treatment with an EZH2 inhibitor if the presence of the mutation in the EZH2 substrate pocket domain is detected in the biological sample. 
     
     
         45 . The method of  claim 29 , wherein the lymphoma is non-Hodgkin lymphoma, follicular lymphoma, or diffuse large B-cell lymphoma.