MUTANT SMOOTHENED AND METHODS OF USING THE SAME

• US 20120039893A1
• Filed: 10/05/2011
• Published: 02/16/2012
• Est. Priority Date: 10/05/2010
• Status: Active Grant

• Current Assignee: Curis, Inc.
• Original Assignee: Genentech, Inc. (Roche Holding AG)
• Inventors: de Sauvage, Frederic J.
• Granted Patent: US 8,481,680 B2
• Time in Patent Office: Days
• US Class Current: 424/139.1
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 14/723 G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor

Abstract

The emergence of mutations in tyrosine kinases following treatment of cancer patients with molecular-targeted therapy represents a major mechanism of acquired drug resistance. Here, we describe a mutation in the serpentine receptor, Smoothened (SMO), which results in resistance to a Hedgehog (Hh) pathway inhibitor in medulloblastoma. A single amino acid substitution in a conserved glutamic acid residue of SMO maintains Hh signaling, but results in the inability of the Hh pathway inhibitor, GDC-0449, to bind SMO and suppress the pathway. The invention provides screening methods to detect SMO mutations and methods to screen for drugs that specifically modulate mutant SMO exhibiting drug resistance.

Claims

1. An isolated nucleic acid molecule encoding a mutant SMO protein comprising an amino acid sequence of that is at least 95% identical to SEQ ID NO:

1 wherein said amino acid sequence comprises an amino acid other than glutamic acid at amino acid 518.

2. The isolated nucleic acid sequence of claim 1 wherein the mutant SMO protein comprises the amino acid sequence of SEQ ID NO:

2 wherein said amino acid sequence comprises a alanine (A) or lysine (K) at amino acid 518.

3. The isolated nucleic acid sequence of claim 1 comprising a parental nucleic acid sequence of SEQ ID NO:

3, wherein said sequence contains a mutation that alters the sequence encoding amino acid 518 to encode a different amino acid.

4. A nucleic acid probe capable of specifically hybridizing to nucleic acid encoding a mutated SMO protein or fragment thereof incorporating a mutation in the sequence encoding amino acid 518.

5. The probe of claim 4 wherein said probe is complementary to said nucleic acid encoding the mutated SMO or said fragment thereof.

6. The probe of claim 4 having a length of about 10 to about 50 nucleotides.

7. The probe of claim 4 further comprising a detectable label.

8. An isolated mutant SMO protein comprising an amino acid sequence of that is at least 95% identical to SEQ ID NO:

2 wherein said amino acid sequence comprises an amino acid other that glutamic acid at amino acid 518.

9. The isolated mutant SMO protein of claim 8 comprising the amino acid sequence of SEQ ID NO:

2 wherein said amino acid sequence comprises a an amino acid other than glutamic acid at amino acid 518.

10. The isolated mutant SMO protein of claim 8 or 9 wherein said amino acid sequence comprises alanine (A) or lysine (K) at amino acid 518.

11. An antibody that specifically binds to the mutant SMO protein of claim 8 or 9, wherein the epitope of said antibody does not bind wild-type SMO having an glutamic acid at position 518.

12. The antibody of claim 11 wherein said antibody is a monoclonal antibody, a chimeric antibody, a humanized antibody, a single chain antibody or an antigen-binding fragment thereof.

13. The antibody of claim 11 wherein said antibody is conjugated to a cytotoxic agent.

14. The antibody of claim 11 wherein said antibody inhibits SMO activity.

15. A method of detecting a mutated SMO gene in a sample comprising amplifying from said sample nucleic acid corresponding to the carboxy-terminus of transmembrane domain 7 of SMO, or a fragment thereof suspected of containing a mutation, and comparing the electrophoretic mobility of the amplified nucleic acid to the electrophoretic mobility of corresponding wild-type SMO gene or fragment thereof.

16. The method of claim 15 wherein the electrophoretic mobility is determined on polyacrylamide gel.

17. A method of identifying at least one SMO mutation in a sample comprising contacting nucleic acid from said sample with a nucleic acid probe that is capable of specifically hybridizing to nucleic acid encoding a mutated SMO protein, or fragment thereof incorporating a mutation that alters the sequence encoding amino acid 518 to an amino acid other than glutamic acid, and detecting said hybridization.

18. The method of claim 17 wherein said probe is detectably labeled.

19. The method of claim 17 wherein said probe is an antisense oligomer.

20. The method of claim 17 wherein the SMO gene or a fragment thereof in said nucleic acid said sample is amplified and contacted with said probe.

21. A method for identifying a tumor in a human subject that is resistant to treatment with GDC-0449 comprising determining the presence of a mutated SMO gene or mutated SMO protein in a sample of said tumor wherein said mutation is located in the SMO gene encoding amino acid 518 whereby the presence of said mutated SMO gene or mutated SMO protein indicates that said tumor is resistant to treatment with a GDC-0449.

22. The method of claim 21 further comprising treating said subject having a tumor that is not susceptible to treatment with GDC-0449 with a compound that binds said mutated SMO.

23. The method of claim 21 wherein the presence or absence of said mutation is performed by examining a nucleic acid sample.

24. The method of claim 21 wherein the presence or absence of said mutation is performed by examining a protein sample.

25. A method of screening for compounds that inhibit signaling of a mutant SMO protein that incorporates a mutation at amino acid 518 comprising contacting said mutant SMO with a test compound and detecting binding of said compound to said mutant SMO whereby binding of said test compound to mutant SMO indicates that said test compound is an inhibitor of mutant SMO.

26. A method of screening for compounds that inhibit signaling of a mutant SMO protein that incorporates a mutation at amino acid 518 comprising contacting a cell that expresses said mutant SMO with a test compound and detecting activity of Gli in said cell whereby the presence of Gli activity indicates that said test compound is not an inhibitor of mutant SMO.

27. A method for treating cancer by administering to a patient in need thereof a compound that specifically binds to a mutant SMO protein having a mutation resulting in an amino acid at position 518 other than glutamic acid.

28. The method of claim 27 wherein said compound is an antibody that specifically binds a mutant SMO having an amino acid other than glutamic acid at position 518.

29. The method of claim 27 wherein said compound is a compound of Formula I, II and/or III.

30. A method of delaying or preventing acquired resistance to SMO inhibitors comprising administering

(a) a SMO inhibitor selected from the group consisting of a compound having the structural formula of Formula I, Formula II and Formula III, and

(b) a PI3K inhibitor to a patient in need thereof.