BRUTON'S TYROSINE KINASE AS ANTI-CANCER DRUG TARGET

• US 20140073593A1
• Filed: 08/20/2013
• Published: 03/13/2014
• Est. Priority Date: 11/07/2008
• Status: Active Grant

• Current Assignee: The Research Foundation for The State University of New York (State University of New York)
• Original Assignee: The Research Foundation for The State University of New York (State University of New York)
• Inventors: Conklin, Douglas S., Eifert, Cheryl, Kourtidis, Antonis
• Granted Patent: US 9,095,592 B2
• Time in Patent Office: Days
• US Class Current: 514/34
• CPC Class Codes: A61K 31/519 ortho- or peri-condensed with heterocyclic rings
• CPC Class Codes: A61K 31/704 attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin digitoxin A61K31/7048
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 16/40 against enzymes
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12Y 207/10001 Receptor protein-tyrosine kinase (2.7.10.1)
• CPC Class Codes: C12Y 207/10002 Non-specific protein-tyrosine kinase (2.7.10.2), i.e. spleen tyrosine kinase
• CPC Class Codes: G01N 2333/9121 with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases
• CPC Class Codes: G01N 33/573 for enzymes or isoenzymes
• CPC Class Codes: G01N 33/57415 of breast

Abstract

Receptor protein kinases (RPTKs) transmit extracellular signals across the plasma membrane to cytosolic proteins, stimulating formation of complexes that regulate key cellular functions. Over half of the known tyrosine kinases are implicated in human cancers and are therefore highly promising drug targets. A large-scale loss-of-function analysis of tyrosine kinases using RNA interference in the clinically relevant Erb-B2 positive, BT474 breast cancer cell line showed that Bruton'"'"'s tyrosine kinase (BTK), a cytosolic, non-receptor tyrosine kinase that has been extensively studied for its role in B cell development, is required, in altered form, for BT474 breast cancer survival. This alternative form contains an amino-terminal extension that is also present in tumorigenic breast cells at significantly higher levels than in normal breast cells.

Claims

1. An interfering double stranded RNA that is at least partially complementary to SEQ ID NO:

2 that inhibits expression of a protein encoded by SEQ ID NO;

2.

2. An interfering double stranded RNA having a sense strand comprising the nucleotide sequence of SEQ ID NO:

66.

3. An interfering double stranded RNA having an antisense strand comprising the nucleotide sequence of SEQ ID NO:

67.

4. An interfering double stranded RNA having a sense strand comprising the nucleotide sequence of SEQ ID NO:

68.

5. An interfering double stranded RNA having an antisense strand comprising the nucleotide sequence of SEQ ID NO:

69.

6. An isolated antibody that specifically binds to the polypeptide of the amino acid sequence set forth in SEQ ID NO:

3.

7. An isolated antibody that specifically binds to the polypeptide of an amino acid sequence at least 95% identical to SEQ ID NO:

3.

8. A method of treating cancer, comprising:

a) providing;

i) subject with breast cancer,

ii) a chemotherapeutic agent, and

iii) an inhibitor of a gene encoding a cytoplasmic tyrosine kinase; and

b) treating said subject with said chemotherapeutic agent and said inhibitor.

9. The method of claim 8, wherein said cytoplasmic tyrosine kinase is Bruton'"'"'s Tyrosine Kinase.

10. The method of claim 8, wherein said cytoplasmic tyrosine kinase is a variant of Bruton'"'"'s Tyrosine Kinase comprising an amino-terminal extension.

11. The method of claim 10, wherein said extension comprises an additional 34 amino acids.

12. The method of claim 8, wherein said inhibitor comprises an interfering double stranded RNA.

13. The method of claim 8, wherein said chemotherapeutic agent comprises Doxorubicin.

14. The method of claim 8, wherein treating with said chemotherapeutic agent and said inhibitor results in reduced proliferation of the breast cancer cells within said subject.

15. The method of claim 12, wherein said interfering double stranded RNA comprises a sense strand having the nucleotide sequence of SEQ ID NO:

66.

16. The method of claim 12, wherein said interfering double stranded RNA comprises an antisense strand having the nucleotide sequence of SEQ ID NO:

67.

17. The method of claim 12, wherein said interfering double stranded RNA comprises a sense strand having the nucleotide sequence of SEQ ID NO:

68.

18. The method of claim 12, wherein said interfering double stranded RNA comprises an antisense strand having the nucleotide sequence of SEQ ID NO:

69.

19. The method of claim 8, wherein said inhibitor comprises a mixture of interfering double stranded RNAs comprising a sense strand having the nucleotide sequence of SEQ ID NOs:

66 and 68 and an antisense strand having the nucleotide sequence of SEQ ID NOs;

67 and 69.

20. The method of claim 8, wherein said chemotherapeutic agent is selected from the group consisting of AC (Adriamycin, cyclophosphamide), TAC (taxotere, AC), ABVD (Adriamycin, bleomycin, vinblastine, dacarbazine), BEACOPP (bleomycin, etoposide, Adriamycin, cyclophosphamide, vincristine, procarbazine, prednisone), BEP (bleomycin, etoposide, platinum agent (cisplatin (Platinol)), CAF (cyclophosphamide, Adriamycin, fluorouracil (5-FU)), CAV (cyclophosphamide, Adriamycin, vincristine), CHOP (cyclophosphamide, Adriamycin, vincristine, prednisone), ChlVPP/EVA (chlorambucil, vincristine, procarbazine, prednisone, etoposide, vinblastine, Adriamycin), CVAD/HyperCVAD (cyclophosphamide, vincristine, Adriamycin, dexamethasone), DT-PACE (dexamethasone, thalidomide, cisplatin or platinol, Adriamycin, cyclophosphamide, etoposide), FAC (5-fluorouracil, Adriamycin, cyclophosphamide), m-BACOD (methotrexate, bleomycin, adriamycin, cyclophosphamide, Oncovin (vincristine), dexamethasone), MACOP-B (methotrexate, leucovorin (folinic acid), adriamycin, cyclophosphamide, Oncovin (vincristine), prednisone, bleomycin), ProMACE-MOPP (methotrexate, Adriamycin, cyclophosphamide, etoposide+MOPP), ProMACE-CytaBOM (prednisone, Adriamycin, cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine, methotrexate, leucovorin), VAD (vincristine, Adriamycin, dexamethasone), Regimen I (vincristine, Adriamycin, etoposide, cyclophosphamide) and VAPEC-B (vincristine, Adriamycin, prednisone, etoposide, cyclophosphamide, bleomycin).

21. A method of treating cancer, comprising:

a) providing;

i) a subject with breast cancer cells, at least some of said breast cancer cells exhibiting resistance to a chemotherapeutic agent, and

ii) an inhibitor of a gene encoding a cytoplasmic tyrosine kinase, and

b) treating said subject with said inhibitor.

22. The method of claim 21, wherein said cytoplasmic tyrosine kinase is Bruton'"'"'s Tyrosine Kinase.

23. The method of claim 21, wherein said cytoplasmic tyrosine kinase is a variant of Bruton'"'"'s Tyrosine Kinase comprising an amino-terminal extension.

24. The method of claim 23, wherein said extension comprises an additional 34 amino acids.

25. The method of claim 21, wherein said inhibitor comprises interfering double stranded RNA.

26. The method of claim 21, wherein treating with said inhibitor results in reduced proliferation of at least some of said breast cancer cells within said subject.

27. The method of claim 25, wherein said interfering double stranded RNA comprises a sense strand having the nucleotide sequence of SEQ ID NO:

66.

28. The method of claim 25, wherein said interfering double stranded RNA comprises an antisense strand having the nucleotide sequence of SEQ ID NO:

67.

29. The method of claim 25, wherein said interfering double stranded RNA comprises a sense strand having the nucleotide sequence of SEQ ID NO:

68.

30. The method of claim 25, wherein said interfering double stranded RNA comprises an antisense strand having the nucleotide sequence of SEQ ID NO:

69.

31. The method of claim 21, wherein said inhibitor comprises a mixture of interfering double stranded RNAs comprising a sense strand having the nucleotide sequence of SEQ ID NOs:

66 and 68 and an antisense strand having the nucleotide sequence of SEQ ID NOs;

67 and 69.

32. The method of claim 21, wherein said inhibitor results in reduced proliferation of at least some breast cancer cells within said subject identified as resistant to said chemotherapeutic agent.

33. The method of claim 21, wherein said chemotherapeutic agent is selected from the group consisting of AC (Adriamycin, cyclophosphamide), TAC (taxotere, AC), ABVD (Adriamycin, bleomycin, vinblastine, dacarbazine), BEACOPP (bleomycin, etoposide, Adriamycin, cyclophosphamide, vincristine, procarbazine, prednisone), BEP (bleomycin, etoposide, platinum agent (cisplatin (Platinol)), CAF (cyclophosphamide, Adriamycin, fluorouracil (5-FU)), CAV (cyclophosphamide, Adriamycin, vincristine), CHOP (cyclophosphamide, Adriamycin, vincristine, prednisone), ChlVPP/EVA (chlorambucil, vincristine, procarbazine, prednisone, etoposide, vinblastine, Adriamycin), CVAD/H erCVAD (cyclophosphamide, vincristine, Adriamycin, dexamethasone), DT-PACE (dexamethasone, thalidomide, cisplatin or platinol, Adriamycin, cyclophosphamide, etoposide), FAC (5-fluorouracil, Adriamycin, cyclophosphamide), m-BACOD (methotrexate, bleomycin, adriamycin, cyclophosphamide, Oncovin (vincristine), dexamethasone), MACOP-B (methotrexate, leucovorin (folinic acid), adriamycin, cyclophosphamide, Oncovin (vincristine), prednisone, bleomycin), ProMACE-MOPP (methotrexate, Adriamycin, cyclophosphamide, etoposide+MOPP), ProMACE-CytaBOM (prednisone, Adriamycin, cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine, methotrexate, leucovorin), VAD (vincristine, Adriamycin, dexamethasone), Regimen I (vincristine, Adriamycin, etoposide, cyclophosphamide) and VAPEC-B (vincristine, Adriamycin, prednisone, etoposide, cyclophosphamide, bleomycin).

34. A method of treating cancer, comprising:

a) providing;

i) a subject with breast cancer,

ii) a chemotherapeutic agent, and

iii) an inhibitor of a gene encoding a cytoplasmic tyrosine kinase,

b) treating said subject with said inhibitor; and

c) after step b), treating said subject with said chemotherapeutic.

35. The method of claim 34, wherein said cytoplasmic tyrosine kinase is Bruton'"'"'s Tyrosine Kinase.

36. The method of claim 34, wherein said cytoplasmic tyrosine kinase is a variant of Bruton'"'"'s Tyrosine Kinase comprising an amino-terminal extension.

37. The method of claim 34, wherein said extension comprises an additional 34 amino acids.

38. The method of claim 34, wherein said inhibitor comprises interfering double stranded RNA.

39. The method of claim 34, wherein said chemotherapeutic agent comprises Doxorubicin.

40. The method of claim 34, wherein treating with said chemotherapeutic agent results in reduced proliferation of the breast cancer cells within said subject.

41. The method of claim 34, wherein said interfering double stranded RNA comprises a sense strand having the nucleotide sequence of SEQ ID NO:

66.

42. The method of claim 38, wherein said interfering double stranded RNA comprises an antisense strand having the nucleotide sequence of SEQ ID NO:

67.

43. The method of claim 38, wherein said interfering double stranded RNA comprises a sense strand having the nucleotide sequence of SEQ ID NO:

68.

44. The method of claim 38, wherein said interfering double stranded RNA comprises an antisense strand having the nucleotide sequence of SEQ ID NO:

69.

45. The method of claim 38, wherein said inhibitor comprises a mixture of interfering double stranded RNAs comprising a sense strand having the nucleotide sequence of SEQ ID NOs:

66 and 68 and an antisense strand having the nucleotide sequence of SEQ ID NOs;

67 and 69.

46. The method of claim 34, wherein said inhibitor results in reduced proliferation of the breast cancer cells within said subject.

47. The method of claim 34, wherein said chemotherapeutic agent is selected from the group consisting of AC (Adriamycin, cyclophosphamide), TAC (taxotere, AC), ABVD (Adriamycin, bleomycin, vinblastine, dacarbazine), BEACOPP (bleomycin, etoposide, Adriamycin, cyclophosphamide, vincristine, procarbazine, prednisone), BEP (bleomycin, etoposide, platinum agent (cisplatin (Platinol)), CAF (cyclophosphamide, Adriamycin, fluorouracil (5-FU)), CAV (cyclophosphamide, Adriamycin, vincristine), CHOP (cyclophosphamide, Adriamycin, vincristine, prednisone), ChlVPP/EVA (chlorambucil, vincristine, procarbazine, prednisone, etoposide, vinblastine, Adriamycin), CVAD/HyperCVAD (cyclophosphamide, vincristine, Adriamycin, dexamethasone), DT-PACE (dexamethasone, thalidomide, cisplatin or platinol, Adriamycin, cyclophosphamide, etoposide), FAC (5-fluorouracil, Adriamycin, cyclophosphamide), m-BACOD (methotrexate, bleomycin, adriamycin, cyclophosphamide, Oncovin (vincristine), dexamethasone), MACOP-B (methotrexate, leucovorin (folinic acid), adriamycin, cyclophosphamide, Oncovin (vincristine), prednisone, bleomycin), ProMACE-MOPP (methotrexate, Adriamycin, cyclophosphamide, etoposide+MOPP), ProMACE-CytaBOM (prednisone, Adriamycin, cyclophosphamide, etoposide, cytarabine, bleomycin, vincristine, methotrexate, leucovorin), VAD (vincristine, Adriamycin, dexamethasone), Regimen I (vincristine, Adriamycin, etoposide, cyclophosphamide) and VAPEC-B (vincristine, Adriamycin, prednisone, etoposide, cyclophosphamide, bleomycin).