METHODS AND COMPOSITIONS RELATING TO THE REGULATION OF APOPTOSIS BY MUC1 AND BH3-CONTAINING PROAPOPTOTIC PROTEINS

• US 20090087437A1
• Filed: 02/01/2008
• Published: 04/02/2009
• Est. Priority Date: 02/02/2007
• Status: Active Grant

• Current Assignee: Dana-Farber Cancer Institute
• Original Assignee: Dana-Farber Cancer Institute
• Inventors: Kufe, Donald W.
• Granted Patent: US 7,871,784 B2
• Time in Patent Office: Days
• US Class Current: 424/139.100
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/06 Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: C07K 14/4747 Apoptosis related proteins
• CPC Class Codes: G01N 2500/02 Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
• CPC Class Codes: G01N 2500/10 involving cells

Abstract

This invention relates to regulation of cell signaling, cell growth, and more particularly to the regulation of cancer or immune cell growth. The invention provides methods of inhibiting interactions between MUC1 and BH3-containing proapoptotic proteins, methods of inhibiting MUC1 expression, and methods of promoting apoptosis. Also provided are screening methods for compounds that inhibit interactions between MUC1 and BH3-containing proapoptotic proteins and pharmaceutical compositions of the same.

Claims

1. A method of identifying a compound that inhibits the binding of MUC1 to a BH3-containing proapoptotic protein, the method comprising:

(a) contacting a MUC1 reagent with a BH3-containing proapoptotic protein reagent in the presence of a candidate compound; and

(b) determining whether the candidate compound inhibits binding of the MUC1 reagent to the BH3-containing proapoptotic reagent.

2. The method of claim 1, wherein the BH3-containing proapoptotic protein reagent comprises BID, t-BID, BAX, BIM, BAK, or a MUC1-binding fragment thereof.

3. The method of claim 1, wherein the MUC1 reagent comprises the cytoplasmic domain of MUC1.

4. The method of claim 3, wherein the cytoplasmic domain of MUC1 has the sequence SEQ ID NO:

2.

9. A compound identified by the method of claim 1.

10. A pharmaceutical composition comprising a compound identified by the method of claim 1.

5. A method of generating a compound that inhibits the interaction between MUC1 and a BH3-containing proapoptotic protein, the method comprising:

(a) providing a three-dimensional structure of a molecule or a molecular complex, the structure comprising;

(i) the cytoplasmic domain of MUC1 or a BH3-containing proapoptotic protein-binding fragment thereof;

(ii) a molecule comprising a BH3-containing proapoptotic protein or a MUC1-binding fragment thereof;

or (iii) a molecular complex comprising (i) and (ii);

(b) designing, based on the three-dimensional structure, a compound comprising a region that inhibits the interaction between MUC1 and the BH3-containing proapoptotic protein; and

(c) producing the compound.

6. The method of claim 5, wherein the BH3-containing proapoptotic protein is BID, t-BID, BAX, BIM, BAK, or a MUC1-binding fragment thereof.

7. The method of claim 5, wherein MUC1 comprises the cytoplasmic domain of MUC1.

8. The method of claim 7, wherein the cytoplasmic domain of MUC1 has the sequence SEQ ID NO:

2.

11. A process of manufacturing a compound, the process comprising:

(a) contacting a MUC1 reagent with a BH3-containing proapoptotic protein reagent in the presence of a candidate compound;

(b) determining whether the candidate compound inhibits binding of the MUC1 reagent to the BH3-containing proapoptotic reagent; and

(c) after determining that a compound inhibits the interaction between MUC1 and a BH3-containing proapoptotic protein, manufacturing the compound.

12. An in vitro method of inhibiting an interaction between MUC1 and a BH3-containing proapoptotic protein, the method comprising contacting (i) a MUC1 reagent;

(ii) a BH3-containing proapoptotic protein reagent;

or (iii) a molecular complex comprising (i) or (ii) with a compound that inhibits the interaction between MUC1 and a BH3-containing proapoptotic protein.

13. The method of claim 12, wherein the BH3-containing proapoptotic protein reagent comprises BID, t-BID, BIM, BAX, BAK, or a MUC1-binding fragment thereof.

14. The method of claim 12, wherein the MUC1 reagent comprises the cytoplasmic domain of MUC1.

15. The method of claim 14, wherein the cytoplasmic domain of MUC1 has the sequence SEQ ID NO:

2.

16. The method of claim 12, wherein the contacting occurs in a cell expressing MUC1.

17. An in vitro method of promoting apoptosis in a cell, the method comprising:

(a) identifying a cell as one expressing a BH2-containing antiapoptotic protein; and

(b) contacting (i) a MUC1 reagent;

(ii) a BH3-containing proapoptotic protein reagent;

or (iii) a molecular complex comprising (i) or (ii) with a compound that inhibits the interaction between MUC1 and a BH3-containing proapoptotic protein, wherein the BH3-containing proapoptotic protein is capable of binding to the BH2-containing antiapoptotic protein.

18. The method of claim 17, wherein the BH3-containing proapoptotic protein reagent comprises BID, t-BID, BIM, BAX, BAK, or a MUC1-binding fragment thereof.

19. The method of claim 17, wherein the MUC1 reagent comprises the cytoplasmic domain of MUC1.

20. The method of claim 19, wherein the cytoplasmic domain of MUC1 has the sequence SEQ ID NO:

2.

21. The method of claim 17, wherein the contacting occurs in a cell expressing MUC1.

22. The method of claim 17 wherein the BH2-containing antiapoptotic protein is Bcl-2 or Bcl-x_L.

23. An in vitro method of promoting apoptosis in a cell, the method comprising identifying a cell as one expressing a BH3-containing proapoptotic protein, and culturing the cell with a compound that inhibits the expression of MUC1.

24. The method of claim 23, wherein the compound inhibits transcription of the MUC1 gene.

25. An in vitro method of promoting apoptosis in a cell, the method comprising identifying a cell as one expressing MUC1, and culturing the cell with an inhibitor of a BH2-containing antiapoptotic protein.

26. An in vivo method of inhibiting an interaction between MUC1 and a BH3-containing proapoptotic protein, the method comprising delivering to a subject a compound that inhibits the interaction between MUC1 and a BH3-containing proapoptotic protein.

27. The method of claim 26, further comprising exposing the subject to one or more additional therapeutic agents.

28. The method of claim 27, wherein the one or more additional therapeutic agents comprise one or more chemotherapeutic agents, one or more forms of ionizing radiation, one or more immunotherapy agents, or one or more forms of hormonal therapy.

29. The method of claim 28, wherein the one or more forms of ionizing radiation are gamma-irradiation, X-irradiation, or beta-irradiation.

30. The method of claim 28, wherein the one or more chemotherapeutic agents are selected from the group consisting of cisplatin, carboplatin, procarbazine, mechlorethamine, cyclophosphamide, camptothecin, adriamycin, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, verampil, podophyllotoxin, tamoxifen, taxol, transplatinum, 5-fluorouracil, vincristin, vinblastin, methotrexate, and an analog of any of the aforementioned.

31. The method of claim 27, wherein the one or more therapeutic agents are inhibitors of HER2.

32. The method of claim 31, wherein the inhibitor of HER2 is Herceptin, Iressa, Tarceva, Erbitux, Lapatinib, or Sutent.

33. The method of claim 27, wherein the one or more therapeutic agents are a non-steroidal anti-inflammatory drug (NSAID), a disease-modifying anti-rheumatic drug (DMARD), a biological response modifier, or a corticosteroid.

34. The method of claim 33, wherein the biological response modifier is an anti-TNF agent.

35. The method of claim 34, wherein the anti-TNF agent comprises a soluble TNF receptor or an antibody specific for TNF.

36. The method of claim 35, wherein the antibody specific for TNF is selected from the group consisting of:

adalimumab, infliximab, or etanercept.

37. The method of claim 26, wherein the compound comprises the cytoplasmic domain of MUC1.

38. The method of claim 37, wherein the cytoplasmic domain of MUC1 comprises the sequence SEQ ID NO:

2.

39. The method of claim 26, wherein the compound comprises a small molecule, an antibody, an antibody fragment, a peptide, or a peptidomimetic.

40. The method of claim 26, wherein the delivery comprises, when the compound is a polypeptide, administering to the subject a nucleic acid comprising a nucleotide sequence encoding the polypeptide, the nucleotide sequence being operably-linked to a transcriptional regulatory sequence.

41. The method of claim 40, wherein the nucleic acid is in a recombinant cell transfected with the nucleic acid and secreting the polypeptide.

42. The method of claim 41, wherein the recombinant cell is a transfected cell, or the progeny of a transfected cell, made by transfecting a cell derived from the subject.

43. The method of claim 26, wherein the subject has, or is suspected of having, a cancer comprising one or more cells expressing MUC1.

44. An in vivo method of promoting apoptosis in a cell, the method comprising identifying a subject as one having a cancer comprising one or more cells expressing a BH3-containing proapoptotic protein, and delivering to the subject a compound that inhibits MUC1.

45. The method of claim 44, wherein the cancer is selected from the group consisting of lung cancer, breast cancer, colon cancer, pancreatic cancer, renal cancer, stomach cancer, liver cancer, bone cancer, hematological cancer, neural tissue cancer, melanoma, thyroid cancer, ovarian cancer, testicular cancer, prostate cancer, cervical cancer, vaginal cancer, and bladder cancer.

46. The method of claim 44, wherein the cancer is a B cell lymphoma.

47. The method of claim 44, wherein the subject is a mammal.

48. The method of claim 47, wherein the mammal is a human.

49. The method of claim 44, wherein the compound inhibits transcription of the MUC1 gene.

50. An in vivo method of promoting apoptosis in a cell, the method comprising identifying a subject as one having a cancer comprising one or more cells expressing a BH2-containing antiapoptotic protein, and delivering to the subject a compound that inhibits the interaction between MUC1 and a BH3-containing proapoptotic protein, wherein the BH3-containing proapoptotic protein is capable of binding to the BH2-containing antiapoptotic protein.

51. The method of claim 50, wherein the BH2-containing antiapoptotic protein is Bcl-2 or Bcl-x_L.

52. An in vivo method of promoting apoptosis in a cell, the method comprising identifying a subject as having a cancer comprising one or more cells expressing MUC1, and delivering to the subject an inhibitor of a BH2-containing antiapoptotic protein.

53. An in vivo method of promoting apoptosis in a cell, the method comprising identifying a subject as one having a inflammatory condition mediated by one or more inflammatory cells expressing a BH3-containing proapoptotic protein, and delivering to the subject a compound that inhibits MUC1.

54. The method of claim 53, wherein the inflammatory condition is an autoimmune disease.

55. The method of claim 53, wherein the autoimmune disease is multiple sclerosis (MS), rheumatoid arthritis (RA), insulin-dependent diabetes mellitus (IDDM), or muscular dystrophy.

56. The method of claim 53, wherein the inflammatory condition is one selected from the group consisting of:

osteoarthritis, spondyloarthropathies, POEMS syndrome, Crohn'"'"'s disease, multicentric Castleman'"'"'s disease, systemic lupus erythematosus, dermatomyositis, polymyositis, Guillain Barre syndrome, Wegener'"'"'s granulomatous, polyarteritis nodosa, polymyalgia rheumatica, temporal arteritis, Sjogren'"'"'s syndrome, Bechet'"'"'s disease, Churg-Strauss syndrome, and Takayasu'"'"'s arteritis.

57. The method of claim 53, wherein the compound inhibits transcription of the MUC1 gene.

58. An in vivo method of promoting apoptosis in a cell, the method comprising:

(a) identifying a subject as one having a inflammatory condition mediated by one or more inflammatory cells expressing a BH2-containing antiapoptotic protein; and

(b) delivering to the subject a compound that inhibits the interaction between MUC1 and a BH3-containing proapoptotic protein, wherein the BH3-containing proapoptotic protein is capable of binding to the BH2-containing antiapoptotic protein.

59. The method of claim 58, wherein the BH2-containing antiapoptotic protein is Bcl-2 or Bcl-x_L.

60. An in vivo method of promoting apoptosis in a cell, the method comprising identifying a subject as one having a inflammatory condition mediated by one or more inflammatory cells expressing MUC1, and delivering to the subject an inhibitor of a BH2-containing antiapoptotic protein.

61. The method of claim 60, wherein the BH2-containing antiapoptotic protein is Bcl-2 or Bcl-x_L.

62. The method of claim 60, wherein the compound inhibits transcription of the MUC1 gene.