Inhibitors of human EZH2 and methods of use thereof

• US 8,691,507 B2
• Filed: 07/23/2013
• Issued: 04/08/2014
• Est. Priority Date: 09/10/2010
• Status: Active Grant

• Current Assignee: Epizyme, Inc. (Ipsen Sa)
• Original Assignee: Epizyme, Inc. (Ipsen Sa)
• Inventors: Copeland, Robert A., Richon, Victoria M., Scott, Margaret D., Sneeringer, Christopher J., Kuntz, Kevin W., Knutson, Sarah K., Pollock, Roy M.
• Primary Examiner(s): Dunston, Jennifer
• Assistant Examiner(s): Leith, Nancy J
• Application Number:
  US13/949,026
• Publication Number:
  US 20130303555A1
• Time in Patent Office: 259 Days
• Field of Search: None
• US Class Current: 435/6.11
• 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 determining the presence of an EZH2 gene mutation in a sample from a subject and 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:

(i) providing a nucleic acid sample from a biological sample obtained from a subject;

(ii) contacting the nucleic acid sample with at least one primer that specifically hybridizes to a portion of SEQ ID NO;

7 having a mutation at the nucleotides encoding position Tyr641 (Y641) of Enhancer of Zeste homolog 2 (EZH2) of SEQ ID NO;

1, wherein the mutation increases EZH2 trimethylation of Lys27 of histone H3 (H3-K27);

(iii) detecting the presence of the mutation at the nucleotides encoding Y641 in the nucleic acid sample by detecting the presence of a nucleic acid encoding the mutation at Y641;

(iv) identifying the subject as a candidate for treatment; and

(v) administering a therapeutically effective amount of an EZH2 inhibitor to the subject identified in step (iv), wherein the EZH2 inhibitor inhibits the conversion of H3-K27 to trimethylated H3-K27.

2. The method of claim 1, wherein the subject has a cancer selected from leukemia, melanoma, and lymphoma, or is at risk of developing a cancer selected from leukemia, melanoma, and lymphoma.

3. The method of claim 2, wherein the lymphoma is selected from the group consisting of Non-Hodgkin'"'"'s lymphoma, follicular lymphoma and diffuse large B-cell lymphoma (DLBCL) of germinal center B cell-like (GCB) subtype.

4. The method of claim 1, where in the EZH2 inhibitor is a small molecule.

10. The method of claim 1 wherein the mutation at the nucleotides encoding position Y641 is selected from the group consisting of Y641F, Y641H, Y641N and Y641S.

5. A method comprising:

(i) providing a nucleic acid sample from a biological sample obtained from a subject;

(ii) contacting the nucleic acid sample with at least one primer that specifically hybridizes to a portion of SEQ ID NO;

7 having a mutation at the nucleotides encoding position Y641 of EZH2 of SEQ ID NO;

1, wherein the mutation increases EZH2 trimethylation of Lys27 of histone H3 (H3-K27);

(iii) detecting the presence of the mutation at the nucleotides encoding Y641 in the nucleic acid sample by detecting the presence of a nucleic acid encoding the mutation at Y641;

(iv) identifying the subject as a candidate for treatment;

(v) selecting a therapy that includes the administration of a therapeutically effective amount of an EZH2 inhibitor to the subject identified in step (iv), wherein the EZH2 inhibitor inhibits the conversion of H3-K27 to trimethylated H3-K27; and

(vi) administering the therapeutically effective amount of the EZH2 inhibitor that inhibits the conversion of H3-K27 to trimethylated H3-K27 to the subject.

6. The method of claim 5, wherein the subject has a cancer selected from leukemia, melanoma, and lymphoma, or is at risk of developing a cancer selected from leukemia, melanoma, and lymphoma.

7. The method of claim 6, wherein the lymphoma is selected from the group consisting of Non-Hodgkin'"'"'s lymphoma, follicular lymphoma and diffuse large B-cell lymphoma (DLBCL) of germinal center B cell-like (GCB) subtype.

8. The method of claim 5, wherein the EZH2 inhibitor is a small molecule.

11. The method of claim 5, wherein the mutation at the nucleotides encoding position Y641 is selected from the group consisting of Y641F, Y641H, Y641N and Y641S.

9. A method comprising,

(i) providing a nucleic acid sample from a biological sample obtained from a subject;

(ii) contacting the nucleic acid sample with at least one primer that specifically hybridizes to a portion of SEQ ID NO;

7 having a mutation at the nucleotides encoding position Y641 of EZH2 of SEQ ID NO;

1, wherein the mutation increases EZH2 trimethylation of Lys27 of histone H3 (H3-K27);

(iii) amplifying the portion of SEQ ID NO;

7 having a mutation at the nucleotides encoding position Y641;

(iv) detecting the mutation at the nucleotides encoding position Y641 by detecting the presence of the amplified nucleic acid;

(v) selecting a therapy that includes the administration of a therapeutically effective amount of an EZH2 inhibitor to the subject when the amplified nucleic acid is detected in step (iv), wherein the EZH2 inhibitor inhibits the conversion of H3-K27 to trimethylated H3-K27; and

(vi) administering the therapeutically effective amount of the EZH2 inhibitor that inhibits the conversion of H3-K27 to trimethylated H3-K27 to the subject.

12. The method of claim 9, wherein the mutation at the nucleotides encoding position Y641 is selected from the group consisting of Y641F, Y641H, Y641N and Y641S.