Diagnostic and Therapeutic Targets for Leukemia

• US 20090061443A1
• Filed: 12/06/2006
• Published: 03/05/2009
• Est. Priority Date: 01/20/2006
• Status: Active Grant

• Current Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Original Assignee: University of North Carolina At Chapel Hill (University of North Carolina System)
• Inventors: Zhang, Yi, Okada, Yuki
• Granted Patent: US 8,524,467 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12Q 1/6886 for cancer immunoassay for cancer G01N33/574
• CPC Class Codes: C12Q 1/6897 involving reporter genes operably linked to promoters
• CPC Class Codes: C12Q 2600/136 Screening for pharmacological compounds
• CPC Class Codes: C12Q 2600/154 Methylation markers
• CPC Class Codes: G01N 33/5011 for testing antineoplastic activity
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention relates to methods of identifying candidate compounds for the treatment of leukemia and diagnostic methods based on histone methylation and HoxA5 promoter activity.

Claims

1. A method of identifying a candidate compound for the prevention and/or treatment of leukemia, the method comprising:

contacting a DOT1L polypeptide with a CALM-AF10 fusion protein in the presence of a test compound under conditions sufficient for binding of the DOT1L polypeptide to the CALM-AF10 fusion protein; and

detecting interaction between the DOT1L polypeptide and the CALM-AF10 fusion protein,

wherein a reduction in interaction between the DOT1L polypeptide and the CALM-AF10 fusion protein in the presence of the test compound as compared with the level of interaction in the absence of the test compound indicates that the test compound is a candidate compound for the prevention and/or treatment of leukemia.

2. The method of claim 1, wherein DOT1L polypeptide interaction with the CALM-AF10 fusion protein is evaluated by determining binding between the DOT1L polypeptide and CALM-AF10.

3. The method of claim 1, wherein DOT1L polypeptide interaction with the CALM-AF10 fusion protein is evaluated by determining nuclear localization of CALM-AF10.

4. The method of claim 1, wherein DOT1L polypeptide interaction with the CALM-AF10 fusion protein is evaluated by determining histone H3 lysine 79 (H3-K79) methylation of HoxA5.

5. The method of claim 4, wherein the H3-K79 methylation of the HoxA5 promoter is determined.

6. The method of claim 1, wherein DOT1L polypeptide interaction with the CALM-AF10 fusion protein is evaluated by determining HoxA5 promoter activity.

7. The method of claim 6, wherein the HoxA5 promoter is operably associated with a heterologous nucleic acid encoding a reporter molecule, and the reporter molecule is detected.

8. The method of claim 7, wherein the reporter molecule is a polypeptide.

9. The method of claim 8, wherein the polypeptide is an enzyme.

10. The method of claim 1, wherein the method is carried out as a cell-free assay.

11. The method of claim 1, wherein the method is carried out as a cell-based assay.

12. The method of claim 1, wherein the DOT1L polypeptide and/or CALM-AF10 fusion protein is expressed from a nucleic acid.

13. The method of claim 1, wherein the leukemia is T cell acute lymphoid leukemia (T-ALL) or acute myeloid leukemia subtype M0/1 (AML-M0/1).

14-18. -18. (canceled)

19. A method of identifying a candidate compound for the prevention and/or treatment of leukemia, the method comprising:

contacting a nucleic acid comprising a HoxA5 promoter region with a CALM polypeptide or CALM-AF10 fusion protein in the presence of a test compound under conditions sufficient for binding of the CALM polypeptide or CALM-AF10 fusion protein to the HoxA5 promoter; and

detecting interaction of the CALM polypeptide or CALM-AF10 fusion protein with the HoxA5 promoter,

wherein a reduction in interaction between the CALM polypeptide or CALM-AF10 fusion protein with the HoxA5 promoter in the presence of the test compound as compared with the level of interaction in the absence of the test compound indicates that the test compound is a candidate compound for the prevention and/or treatment of leukemia.

20. The method of claim 19, wherein the nucleic acid further comprises exon 1 of the HoxA5 gene.

21. The method of claim 19, wherein the nucleic acid further comprises the intron between exons 1 and 2 of the HoxA5 gene.

22. The method of claim 19, wherein the nucleic acid further comprises exon 2 of the HoxA5 gene.

23. The method of claim 19, wherein CALM polypeptide or CALM-AF10 fusion protein interaction with the HoxA5 promoter is evaluated by determining binding of the CALM polypeptide or CALM-AF10 fusion protein to the HoxA5 promoter.

24. The method of claim 19, wherein CALM or CALM-AF10 interaction with the HoxA5 promoter is evaluated by determining histone H3 lysine 79 (H3-K79) methylation of HoxA5.

25. The method of claim 19, wherein CALM or CALM-AF10 interaction with the HoxA5 promoter is evaluated by determining HoxA5 promoter activity.

26. The method of claim 25, wherein the HoxA5 promoter is operatively associated with a nucleotide sequence encoding a reporter molecule and HoxA5 promoter activity is determined by detecting the reporter molecule.

33. The method of claim 19, the method comprising:

contacting a nucleic acid comprising a HoxA5 promoter region with a DOT1L polypeptide and a CALM-AF10 fusion protein in the presence of a test compound under conditions sufficient for binding of the DOT1L polypeptide and the CALM-AF10 fusion protein to form a complex and for the complex to bind to the HoxA5 promoter; and

detecting interaction of the DOT1L/CALM-AF10 complex with the HoxA5 promoter,

wherein a reduction in interaction of the DOT1L/CALM-AF10 complex with the HoxA5 promoter in the presence of the test compound as compared with the level of interaction in the absence of the test compound indicates that the test compound is a candidate compound for the prevention and/or treatment of leukemia.

27-32. -32. (canceled)

34-50. -50. (canceled)

51. A method of identifying a candidate compound for the prevention and/or treatment of leukemia, the method comprising:

contacting a nucleic acid comprising a HoxA5 promoter region with a test compound under conditions sufficient for HoxA5 promoter activity; and

detecting HoxA5 promoter activity,

wherein a reduction in HoxA5 promoter activity in the presence of the test compound as compared with the level of HoxA5 promoter activity in the absence of the test compound indicates that the test compound is a candidate compound for the prevention and/or treatment of leukemia.

52-59. -59. (canceled)

60. A method of identifying a candidate compound for the prevention and/or treatment of T cell acute lymphoid leukemia (T-ALL) or acute myeloid leukemia subtype M0/1 (AML-M0/1):

contacting a DOT1L polypeptide with a histone or nucleosome substrate comprising histone H3 in the presence of a test compound;

detecting histone H3 lysine 79 (H3-K79) methylation of the substrate under conditions sufficient to provide H3-K79 methylation;

wherein an reduction in H3-K79 methylation in the presence of the test compound as compared with the level of H3-K79 methylation in the absence of the test compound indicates that the test compound is a candidate compound for the prevention and/or treatment of T-ALL or AML subtypes M0/1.

61. A method of identifying a candidate compound for the prevention and/or treatment of T cell acute lymphoid leukemia (T-ALL) or acute myeloid leukemia subtype M0/1 (AML-M0/1):

contacting a DOT1L polypeptide with AF10 under conditions sufficient for binding of the DOT1L polypeptide to AF10;

detecting interaction between the DOT1L polypeptide and AF10;

wherein a reduction in interaction between DOT1L polypeptide and AF10 in the presence of the test compound as compared with the level of binding in the absence of the test compound indicates that the test compound is a candidate compound for the prevention and/or treatment of T-ALL or AML subtypes M0/1.

62. A method of diagnosing whether a subject has or is at risk for developing leukemia and/or determining the prognosis for the course of the disease, the method comprising:

obtaining a biological sample comprising nucleosomes from a subject;

detecting histone H3 lysine 79 (H3-K79) methylation associated with the HoxA5 gene;

wherein an increase in HoxA5-associated H3-K79 methylation in the biological sample as compared with the level of HoxA5-associated H3-K79 methylation in a non-leukemic biological sample is diagnostic that the subject has or is at risk of developing leukemia and/or is prognostic of the course of the disease in the subject.

63-68. -68. (canceled)

69. A method of diagnosing whether a subject has or is at risk for developing leukemia and/or determining the prognosis for the course of the disease, the method comprising determining HoxA5 promoter activity in the subject.

70. (canceled)