Targeting deregulated Wnt signaling in cancer using stabilized alpha-helices of BCL-9

1. A method of treating cancer mediated by BCL9/β-catenin binding in a human subject, the method comprising administering to the human subject a therapeutically effective amount of a structurally constrained peptide of an HD2 domain of BCL9 (BCL9-HD2) comprising at least one hydrocarbon staple, wherein the at least one hydrocarbon staple links the side chain of a first non-natural amino acid that replaces a first BCL9-HD2 amino acid selected from the group consisting of Leu-351, Ser-352, Gln-353, Glu-354, Gln-355, His-358, Arg-359, Arg-361, Ser-362, Leu-363, Thr-365, Leu-366, Ile-369, Gln-370, Met-372, Leu-373, and Phe-374 with the side chain of a second non-natural amino acid that replaces a second BCL9-HD2 amino acid selected from the group consisting of Leu-351, Ser-352, Gln-353, Glu-354, Gln-355, Leu-356, Glu-357, His-358, Arg-359, Glu-360, Arg-361, Ser-362, Leu-363, Thr-365, Leu-366, Arg-367, Asp-368, Ile-369, Gln-370, Arg-371, Met-372, Leu-373, and Phe-374,

wherein the first and second non-natural amino acids are two, three, or six amino acids apart, and

wherein the cancer is selected from the group consisting of colorectal cancer, colon cancer, and multiple myeloma.

2. The method of claim 1 , wherein the human subject has been previously identified as in need of a canonical Wnt/β-catenin signaling inhibitor to treat the cancer.

3. The method of claim 1 , wherein the human subject is administered with an additional therapeutic agent, radiation, or chemotherapy.

4. The method of claim 3 , wherein the additional therapeutic agent is an anti-cancer compound.

5. The method of claim 3 , wherein the structurally constrained peptide and the additional therapeutic agent are administered simultaneously or sequentially.

6. The method of claim 1 , wherein the structurally constrained peptide comprises an interacting face comprised of amino acids that interact with β-catenin, wherein the interacting face comprises BCL9 residues Gln-355, His-358, Arg-359, Ser-362, Leu-363, Leu-366, Ile-369, Gln-370, Leu-373, and Phe-374.

7. The method of claim 6 , wherein the interacting face represents a single face of an α-helix.

8. The method of claim 7 , wherein the single face of a helix comprises one, two, three, or four adjacent stacked columns of amino acids, wherein the stacked columns of amino acids are defined by positions a, d, and g; positions b and e; or positions c and f; in an alpha helix having 3.6 amino acids per turn wherein the amino acids are consecutively and serially assigned positions a-g; and positions a and d; positions b and e; or positions c and f in a 3 10 helix having 3 amino acids per turn wherein the amino acids are consecutively and serially assigned positions a-f; or homologues thereof.

9. The method of claim 1 , wherein the at least one hydrocarbon staple is formed by an olefin metathesis reaction.

10. The method of claim 1 , wherein the first and second non-natural amino acids are selected from the following:

11. The method of claim 1 , wherein the structurally constrained peptide comprises 1 to 2 staples within the BCL9 HD2 peptide.

12. The method of claim 1 , wherein the at least one hydrocarbon staple is located at any of the staple positions in any one of the following BCL9 HD2 stapled peptides:

i, i + 4 single staples:

(SEQ ID NO: 8)

X SQE X LEHRERSLQTLRDIQRBLF

(SEQ ID NO: 9)

L X QEQ X EHRERSLQTLRDIQRBLF

(SEQ ID NO: 10)

LS X EQL X HRERSLQTLRDIQRBLF

(SEQ ID NO: 11)

LSQ X QLE X RERSLQTLRDIQRBLF

(SEQ ID NO: 12)

LSQE X LEH X ERSLQTLRDIQRBLF

(SEQ ID NO: 13)

LSQEQ X EHR X RSLQTLRDIQRBLF

(SEQ ID NO: 14)

LSQEQL X HRE X SLQTLRDIQRBLF

(SEQ ID NO: 15)

LSQEQLE X RER X LQTLRDIQRBLF

(SEQ ID NO: 16)

LSQEQLEH X ERS X QTLRDIQRBLF

(SEQ ID NO: 17)

LSQEQLEHR X RSL X TLRDIQRBLF

(SEQ ID NO: 18)

LSQEQLEHRE X SLQ X LRDIQRBLF

(SEQ ID NO: 19)

LSQEQLEHRER X LQT X RDIQRBLF

(SEQ ID NO: 20)

LSQEQLEHRERS X QTL X DIQRBLF

(SEQ ID NO: 21)

LSQEQLEHRERSL X TLR X IQRBLF

(SEQ ID NO: 22)

LSQEQLEHRERSLQ X LRD X QRBLF

(SEQ ID NO: 23)

LSQEQLEHRERSLQT X RDI X RBLF

(SEQ ID NO: 24)

LSQEQLEHRERSLQTL X DIQ X BLF

(SEQ ID NO: 25)

LSQEQLEHRERSLQTLR X IQR X LF

(SEQ ID NO: 26)

LSQEQLEHRERSLQTLRD X QRB X F

(SEQ ID NO: 27)

LSQEQLEHRERSLQTLRDI X RBL X

i, i + 7 staples:

(SEQ ID NO: 28)

X SQEQLE X RERSLQTLRDIQRBLF

(SEQ ID NO: 29)

L X QEQLEH X ERSLQTLRDIQRBLF

(SEQ ID NO: 30)

LS X EQLEHR X RSLQTLRDIQRBLF

(SEQ ID NO: 31)

LSQ X QLEHRE X SLQTLRDIQRBLF

(SEQ ID NO: 32)

LSQE X LEHRER X LQTLRDIQRBLF

(SEQ ID NO: 33)

LSQEQ X EHRERS X QTLRDIQRBLF

(SEQ ID NO: 34)

LSQEQL X HRERSL X TLRDIQRBLF

(SEQ ID NO: 35)

LSQEQLE X RERSLQ X LRDIQRBLF

(SEQ ID NO: 36)

LSQEQLEH X ERSLQT X RDIQRBLF

(SEQ ID NO: 37)

LSQEQLEHR X RSLQTL X DIQRBLF

(SEQ ID NO: 38)

LSQEQLEHRE X SLQTLR X IQRBLF

(SEQ ID NO: 39)

LSQEQLEHRER X LQTLRD X QRBLF

(SEQ ID NO: 40)

LSQEQLEHRERS X QTLRDI X RBLF

(SEQ ID NO: 41)

LSQEQLEHRERSL X TLRDIQ X BLF

(SEQ ID NO: 42)

LSQEQLEHRERSLQ X LRDIQR X LF

(SEQ ID NO: 43)

LSQEQLEHRERSLQT X RDIQRB X F

(SEQ ID NO: 44)

LSQEQLEHRERSLQTL X DIQRBL X

i, i + 3 single staples:

(SEQ ID NO: 45)

X SQ X QLEHRERSLQTLRDIQRBLF

(SEQ ID NO: 46)

L X QE X LEHRERSLQTLRDIQRBLF

(SEQ ID NO: 47)

LS X EQ X EHRERSLQTLRDIQRBLF

(SEQ ID NO: 48)

LSQE X LE X RERSLQTLRDIQRBLF

(SEQ ID NO: 49)

LSQEQ X EH X ERSLQTLRDIQRBLF

(SEQ ID NO: 50)

LSQEQL X HR X RSLQTLRDIQRBLF

(SEQ ID NO: 51)

LSQEQLE X RE X SLQTLRDIQRBLF

(SEQ ID NO: 52)

LSQEQLEH X ER X LQTLRDIQRBLF

(SEQ ID NO: 53)

LSQEQLEHR X RS X QTLRDIQRBLF

(SEQ ID NO: 54)

LSQEQLEHRE X SL X TLRDIQRBLF

(SEQ ID NO: 55)

LSQEQLEHRER X LQ X LRDIQRBLF

(SEQ ID NO: 56)

LSQEQLEHRERS X QT X RDIQRBLF

(SEQ ID NO: 57)

LSQEQLEHRERSL X TL X DIQRBLF

(SEQ ID NO: 58)

LSQEQLEHRERSLQ X LR X IQRBLF

(SEQ ID NO: 59)

LSQEQLEHRERSLQT X RD X QRBLF

(SEQ ID NO: 60)

LSQEQLEHRERSLQTL X DI X RBLF

(SEQ ID NO: 61)

LSQEQLEHRERSLQTLR X IQ X BLF

(SEQ ID NO: 62)

LSQEQLEHRERSLQTLRD X QR X LF

(SEQ ID NO: 63)

LSQEQLEHRERSLQTLRDI X RB X F

(SEQ ID NO: 64)

LSQEQLEHRERSLQTLRDIQ X BL X .

13. The method of claim 1 , wherein the structurally constrained peptide of BCL9-HD2 comprises two hydrocarbon staples and the two hydrocarbon staples are located at any of the positions within any one of the following BCL9 HD2 stapled peptides:

i, i + 3 double staples:

(SEQ ID NO: 65)

X SQ X QLEHRERSLQTLRDIQ X BL X

(SEQ ID NO: 66)

X SQ X QLEHRERSLQTLRDI X RB X F

(SEQ ID NO: 67)

X SQ X QLEHRERSLQTLRD X QRB X F

i, i + 4 double staples:

(SEQ ID NO: 68)

X SQE X LEHRERSLQTLRDI X RBL X

(SEQ ID NO: 69)

X SQE X LEHRERSLQTLRD X QRB X F

(SEQ ID NO: 70)

X SQE X LEHRERSLQTL X DIQR X LF

i, i + 7 double staples:

(SEQ ID NO: 71)

X SQEQLE X RERSLQTL X DIQRBL X

(SEQ ID NO: 72)

X SQEQLE X RERSLQT X RDIQRB X F

(SEQ ID NO: 73)

X SQEQLE X RERSLQ X LRDIQR X LF.

14. The method of claim 1 , wherein the at least one hydrocarbon staple is located at any of the positions within any one of the following BCL9 HD2 stapled peptides:

Mixed i, i + 4; i, i + 3; and i, i + 7 

double staples:

(SEQ ID NO: 74)

X SQE X LEHRERSLQTL X DIQRBL X

(SEQ ID NO: 75)

X SQE X LEHRERSLQT X RDIQRB X F

(SEQ ID NO: 76)

X SQE X LEHRERSLQ X LRDIQR X LF

(SEQ ID NO: 77)

X SQE X LEHRERSLQTLRDIQ X BL X

(SEQ ID NO: 78)

X SQE X LEHRERSLQTLRDI X RB X F

(SEQ ID NO: 79)

X SQE X LEHRERSLQTLRD X QR X LF

(SEQ ID NO: 80)

X SQEQLE X RERSLQTLRDI X RBL X

(SEQ ID NO: 81)

X SQEQLE X RERSLQTLRD X QRB X F

(SEQ ID NO: 82)

X SQEQLE X RERSLQTLR X IQR X LF

(SEQ ID NO: 83)

X SQEQLE X RERSLQTLRDIQ X BL X

(SEQ ID NO: 84)

X SQEQLE X RERSLQTLRDI X RB X F

(SEQ ID NO: 85)

X SQEQLE X RERSLQTLRD X QR X LF

(SEQ ID NO: 86)

X SQ X QLEHRERSLQTLRDI X RBL X

(SEQ ID NO: 87)

X SQ X QLEHRERSLQTLRD X QRB X F

(SEQ ID NO: 88)

X SQ X QLEHRERSLQTLR X IQR X LF

(SEQ ID NO: 89)

X SQ X QLEHRERSLQTL X DIQRBL X

(SEQ ID NO: 90)

X SQ X QLEHRERSLQT X RDIQRB X F

(SEQ ID NO: 91)

X SQ X QLEHRERSLQ X LRDIQR X LF

Sequential i, i + 4 staples:

(SEQ ID NO: 92)

X SQE X LEH X ERSLQTLRDIQRBLF

(SEQ ID NO: 93)

L X QEQ X EHR X RSLQTLRDIQRBLF

(SEQ ID NO: 94)

LS X EQL X HRE X SLQTLRDIQRBLF

(SEQ ID NO: 95)

LSQ X QLE X RER X LQTLRDIQRBLF

(SEQ ID NO: 96)

LSQE X LEH X ERS X QTLRDIQRBLF

(SEQ ID NO: 97)

LSQEQ X EHR X RSL X TLRDIQRBLF

(SEQ ID NO: 98)

LSQEQL X HRE X SLQ X LRDIQRBLF

(SEQ ID NO: 99)

LSQEQLE X RER X LQT X RDIQRBLF

(SEQ ID NO: 100)

LSQEQLEH X ERS X QTL X DIQRBLF

(SEQ ID NO: 101)

LSQEQLEHR X RSL X TLR X IQRBLF

(SEQ ID NO: 102)

LSQEQLEHRE X SLQ X LRD X QRBLF

(SEQ ID NO: 103)

LSQEQLEHRER X LQT X RDI X RBLF

(SEQ ID NO: 104)

LSQEQLEHRERS X QTL X DIQ X BLF

(SEQ ID NO: 105)

LSQEQLEHRERSL X TLR X IQR X LF

(SEQ ID NO: 106)

LSQEQLEHRERSLQ X LRD X QRB X F

(SEQ ID NO: 107)

LSQEQLEHRERSLQT X RDI X RBL X

Sequential i, i + 3 staples:

(SEQ ID NO: 108)

X SQ X QL X HRERSLQTLRDIQRBLF

Sequential i, i + 7 staples:

(SEQ ID NO: 109)

X SQEQLE X RERSLQ X LRDIQRBLF

Mixed sequential staples:

(SEQ ID NO: 110)

X SQ X QLE X RERSLQTLRDIQRBLF

(SEQ ID NO: 111)

X SQ X QLEHRE X SLQTLRDIQRBLF

(SEQ ID NO: 112)

X SQE X LE X RERSLQTLRDIQRBLF

(SEQ ID NO: 113)

X SQE X LEHRER X LQTLRDIQRBLF

(SEQ ID NO: 114)

X SQEQLE X RE X SLQTLRDIQRBLF

(SEQ ID NO: 115)

X SQEQLE X RER X LQTLRDIQRBLF.

15. The method of claim 1 , wherein the structurally constrained peptide comprises an amino acid sequence selected from the group consisting of:

SAH-BCL9 A :

(SEQ ID NO: 3)

LSQEQLEHRERSLQTLR X IQR X LF;

SAH-BCL9 B :

(SEQ ID NO: 4)

LSQEQLEHRERSL X TLR X IQRBLF;

and

SAH-BCL9 C :

(SEQ ID NO: 5)

LSQEQLEHRE X SLQ X LRDIQRBLF.

16. The method of claim 15 , wherein the structurally constrained peptide comprises the amino acid sequence of SEQ ID NO: 3.

17. The method of claim 15 , wherein the structurally constrained peptide comprises the amino acid sequence of SEQ ID NO: 4.