Inhibitors of Human EZH2 and Methods of Use Thereof

• US 20120071418A1
• Filed: 09/12/2011
• Published: 03/22/2012
• Est. Priority Date: 09/10/2010
• Status: Active Grant

• Current Assignee: Epizyme, Inc. (Ipsen Sa)
• Original Assignee: Epizyme, Inc. (Ipsen Sa)
• Inventors: Richon, Victoria M., Pollock, Roy M., Copeland, Robert A., Scott, Margaret D., Sneeringer, Christopher J., Kuntz, Kevin W., Knutson, Sarah K.
• Granted Patent: US 8,895,245 B2
• Time in Patent Office: Days
• US Class Current: 514/19.3
• CPC Class Codes: A61K 31/7076 containing purines, e.g. adenosine, adenylic acid
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07D 473/34 attached in position 6, e.g. adenine
• CPC Class Codes: C07D 493/04 Ortho-condensed systems
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: G01N 2333/91011 Methyltransferases (general) (2.1.1.)
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: G01N 33/57426 leukemia

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.

Claims

1. A method, comprising

a) obtaining a tissue sample from a subject;

b) detecting a dimethylation (me2) level of H3-K27 if present in said tissue sample and comparing said dimethylation (me2) level to a control dimethylation (me2) level;

c) optionally detecting a trimethylation (me3) level of H3-K27 if present in said tissue sample and comparing said trimethylation (me3) level to a control trimethylation (me3) level,

wherein said subject is responsive to an EZH2 inhibitor when said dimethylation (me2) level is absent or lower than said control dimethylation (me2) level or when said trimethylation (me3) level is same as or higher than said control trimethylation (me3) level and said dimethylation (me2) level is absent or lower than said control dimethylation (me2) level.

2. The method of claim 1, further comprising

d) obtaining a ratio of the dimethylation (me2) level to the trimethylation (me3) level of H3-K27 in said tissue sample to obtain a test ratio and comparing the test ratio to a control ratio;

wherein said subject is responsive to an EZH2 inhibitor when said test ratio is lower than said control ratio.

3. The method of claim 1, wherein said subject has a cancer.

4. The method of claim 1, wherein said detecting is performed by an assay selected from Western Blot analysis, immunohistochemistry (IHC), immunofluorescence (IF) and Mass spectrometry (MS).

5. The method of claim 1, wherein said detecting comprises contacting said tissue sample with an agent that binds specifically to dimethylated H3-K27 and/or trimethylated H3-K27.

6. The method of claim 5, wherein said agent is an antibody, a polypeptide, an aptamer or a fragment thereof.

7. A method of treating cancer in a subject in need thereof, comprising

administering to a subject that is has been determined to be responsive to an EZH2 inhibitor by the method of claim 1.

8. The method of claim 3, wherein said cancer is selected from follicular lymphoma and diffuse large B-cell lymphoma (DLBCL).

9. The method of claim 1, wherein said subject expresses a Y641 mutant EZH2 polypeptide.

10. The method of claim 9, wherein said Y641 mutant is selected from Y641F, Y641H, Y641N and Y641S.

20. The method claim 1, wherein the inhibitor of EZH2 is S-adenosyl-L-homocysteine,

11. A method, comprising

administering to a subject expressing a Y641 mutant of an EZH2 polypeptide a therapeutically effective amount of an inhibitor of EZH2.

14. The method of claim 11, wherein the inhibitor inhibits histone methyltransferase activity of the Y641 mutant EZH2 polypeptide.

15. The method of claim 14, wherein the inhibition is selective inhibition.

16. The method of claim 11, wherein the Y641 mutant of the EZH2 polypeptide has a mutation selected from the group consisting of Y641F, Y641H, Y641N, and Y641S.

17. The method of claim 11, wherein said detecting the Y641 mutant of EZH2 is by:

a. whole-genome resequencing,

b. target region resequencing that detects a nucleic acid encoding the Y641 mutant of the EZH2 polypeptide,

c. an antibody that binds specifically to a polypeptide or fragment thereof characteristic of the Y641 mutant of the EZH2 polypeptide;

or

d. a nucleic acid probe that hybridizes to a nucleic acid encoding a polypeptide or fragment thereof characteristic of the Y641 mutant of the EZH2 polypeptide.

18. The method of claim 11, wherein the subject has cancer.

19. The method of claim 18, wherein the cancer is selected from the group consisting of follicular lymphoma and diffuse large B-cell lymphoma (DLBCL).

12. A method, comprising

contacting a cell expressing a Y641 mutant of an EZH2 polypeptide with an effective amount of an inhibitor of EZH2.

13. A method, comprising;

detecting a Y641 mutant of an EZH2 polypeptide if present in a sample from a subject;

wherein the presence of the Y641 mutant indicates the subject is a candidate for treatment with an inhibitor of EZH2.

21. A compound

22. A pharmaceutical composition comprising the compound of claim 21.

23. Use of

24. A method, comprising

combining an isolated Y641 mutant of a EZH2 polypeptide with a histone substrate, a methyl group donor, and a test compound, wherein the histone substrate comprises a form of H3-K27 selected from the group consisting of unmethylated H3-K27, monomethylated H3-K27, dimethylated H3-K27, and any combination of thereof to form a test mixture;

and

detecting methylation of H3-K27 in the histone substrate, thereby identifying the test compound as an inhibitor of the Y641 mutant of EZH2 when methylation of H3-K27 in the presence of the test compound is less than methylation of H3-K27 in the absence of the test compound.

25. The method of claim 24 further comprising

detecting formation of trimethylated H3-K27 in the histone substrate, thereby identifying the test compound as an inhibitor of the Y641 mutant of EZH2 when formation of trimethylated H3-K27 in the presence of the test compound is less than formation of trimethylated H3-K27 in the absence of the test compound.

26. The method of claim 25, further comprising

combining an isolated wild-type EZH2 with a histone substrate, a methyl group donor, and a test compound, wherein the histone substrate comprises a form of H3-K27 selected from the group consisting of monomethylated H3-K27, dimethylated H3-K27, and a combination of monomethylated H3-K27 and dimethylated H3-K27, thereby forming a control mixture;

detecting trimethylation of the histone substrate in each of the test mixture and the control mixture;

calculating the ratio of (a) trimethylation with the Y641 mutant of EZH2 and the test compound (M+) to (b) trimethylation with the Y641 mutant of EZH2 without the test compound (M−

);

calculating the ratio of (c) trimethylation with wild-type EZH2 and the test compound (WT+) to (d) trimethylation with wild-type EZH2 without the test compound (WT−

);

comparing the ratio (a)/(b) with the ratio (c)/(d); and

identifying the test compound as a selective inhibitor of the Y641 mutant of EZH2 when the ratio (a)/(b) is less than the ratio (c)/(d).

27. The method of claim 24, wherein the Y641 mutant of the EZH2 polypeptide has a mutation selected from the group consisting of Y641F, Y641H, Y641N, and Y641S.

28. The method of claim 24, wherein said detecting comprises measuring incorporation of labeled methyl groups.

29. The method of claim 28, wherein the labeled methyl groups are isotopically labeled methyl groups.

30. The method of claim 24, wherein said detecting comprises contacting the histone substrate with an antibody that binds specifically to trimethylated H3-K27.