MODIFIED BCL9 MIMETIC PEPTIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U S. Provi sional Patent

Application No. 62/870,938, filed on July 5, 2019.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on July 6, 2020, is named Sapience_004_WOl_SL.txt and is 49,081 bytes in size.

BACKGROUND

[0003] B-celi CLL/lymphoma 9 (BCL9) is a protein that acts as a co-activator for b- catenin-mediated transcription. BCL9 is over-expressed in many tumors and enhances b- catenin signaling in cancer cells, but not in normal cells from which the tumors originate (Zhan et al. 2017). BCL9 interacts with b-catenin via its a-helical homology domain-2 (HD2). Previous studies have shown that disruption of the BCL9/]3-catenin interaction using hydrocarbon-stapled BCL9 peptides suppresses transcription of Wnt target genes regulating proliferation, migration, invasion, and the metastatic potential of tumor cells (Takada et al, 2012; WO 2017/062518)

SUMMARY OF THE INVENTION

[0004] Some of the main aspects of the present invention are summarized below.

Additional aspects are described in the Detailed Description of the Invention, Examples, Drawings, and Claims sections of this disclosure. The description in each section of this disclosure is intended to be read in conjunction with the other sections. Furthermore, the various embodiments described in each section of this disclosure can be combined in various different ways, and all such combinations are intended to fall within the scope of the present invention.

[0005] The invention provides BCL9 mimetic peptides comprising a modified BCL9 a- helical homology domain-2 (HD2) region. In one embodiment, the invention provides a BCL9 mimetic peptide comprising a modified BCL9 a-helical homology domain-2 (HD2) region, wherein the modified BCL9 HD2 region comprises a variant of the amino acid sequence L S QEQ LEHRER SLQT LRDI Q RMLF (SEQ ID NO: 1), wherein the variant is modified at one or more positions of SEQ ID NO: 1 as follows: (i) E7 is substituted with R; (ii) R1 l is substituted with E; (iii) S12 is substituted with A; (iv) Q14 is substituted with A or E; (v) T15 is substituted with A; (vi) D18 is substituted with A or R; (vii) 119 is substituted with L; (viii) R21 is substituted with E; (ix) M22 is substituted with A or L; (x) W, 1 -Na1, or 2-Nal is added at position 25. The BCL9 mimetic peptide can additionally comprise a modification wherein F24 is substituted with W, I-Nal, or 2-Nal and/or wherein between 1 and 15 consecutive amino acids of SEQ ID NO: 1 are truncated beginning at LI. In one embodiment, the modified BCL9 HD2 region comprises an amino acid sequence selected from the group consisting of: LSQEQLEHRERSLATLRAIQRMLF (SEQ ID NO: 3);

L S QEQLRHREE SLETLRRIQEMLF (SEQ ID NO: 4);

LSQEQLEHRERALQALRAIQRALF (SEQ ID NO: 5); and ALQALR AIQRALF (SEQ ID NO: 6). Also included are retro-inverso BCL9 mimetic peptides comprising D-amino acids in a reversed amino acid sequence relative to an amino acid sequence disclosed herein.

[0006] One embodiment of the invention is a BCL9 mimetic peptide comprising a

modified BCL9 a-helical homology domain-2 (HD2) region, wherein the modified BCL9 HD2 region is a D-amino acid sequence comprising a variant of the D-amino acid sequence FLMRQIDRLTQLS (SEQ ID NO: 7), wherein the variant is modified at one or more positions of SEQ ID NO: 7 as follows: (i) FI is substituted with L or W; (ii) M3 is substituted with A, E, L, or V; (iii) R4 is substituted with O (ornithine); (iv) 16 is substituted with L; (v) D7 is substituted with A or E; (vi) R8 is substituted with A; (vii) T10 is substituted with A, K, Q, or R; (viii) Q11 is substituted with A, K, or R; (ix) S13 is substituted with A. In a particular embodiment, the BCL9 mimetic peptide further comprises W, F, R, 1 -Nal, or 2-Nal, in either D- or L- form, at the N-terminus of the peptide.

[0007] In certain embodiments, the modified BCL9 HD2 region comprises a D-amino acid sequence selected from the group consisting of: FLMRQIDRLTQLA (SEQ ID NO: 8); FLMRQLDRL T QL A (SEQ ID NO: 9); F L A R Q L A R L A Q L A (SEQ ID NO: 10);

WLARQLARLAQLA (SEQ ID NO: 11); WWLARQLARLAQLA (SEQ ID NO: 12);

FLMEQLRRLTELA (SEQ ID NO: 13); FLAEQLRRLAELA (SEQ ID NO: 14);

WLAEQLRRLAELA (SEQ ID NO: 15); WWLARQLERLAQLA (SEQ ID NO: 16); 1-Nal-

[0008] In certain embodiments, the BCL9 mimetic peptides of the invention comprise a modified BCL9 a-helical homology domain-2 (HD2) region of mixed chirality. In a particular embodiment, the modified BCL9 HD2 region comprises an amino acid sequence selected from the group consisting of: (i) F _D R _L [WLARQLARLAQLA] _D (SEQ ID NO: 103);

subscripts denote chirality of the amino acids.

[0009] In some embodiments, the BCL9 mimetic peptide comprises a cell-penetrating region, wherein the BCL9 mimetic peptide is a cell-penetrating peptide. In certain embodiments, the cell-penetrating region has an amino acid sequence selected from the group consisting of or

the cell-penetrating region has a D-amino acid sequence selected from the group consisting [0010] In some embodiments, the BCL9 mimetic peptide comprises an N-terminal group selected from the group consisting of acetyl, naphthyl, octanoyl, phenyl, and isovaleryl, and/or the BCL9 mimetic peptide comprises a C-temiinal amide group.

[0011] In one aspect, BCL9 mimetic peptides of the invention are for use in inhibiting proliferation of and/or promoting cytotoxicity in a neoplastic cell.

[0012] Further aspects of the invention provide a composition comprising a BCL9

mimetic peptide of the invention, for example, a pharmaceutical composition; a kit comprising a BCL9 mimetic peptide of the invention; and a nucleic acid molecule encoding a BCL9 mimetic peptide of the invention.

[0013] The invention additionally provides methods of inhibiting proliferation of and/or promoting cytotoxicity in a neoplastic cell, the methods comprising contacting the neoplastic cell with a BCL9 mimetic peptide of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 shows that modified BCL9 mimetic peptides of the invention antagonize b-catenin and exert anti-proliferative activity in cultured MCF7 breast cancer cells.

[0015] FIG. 2A-2B show that retro inverso BCL9 mimetic peptides of the invention display anti-proliferative activity in cultured MCF7 breast cancer cells. Cytotoxicity data is shown for peptide BCL-26 (FIG. 2A) and peptide BCL-27 (FIG. 2B).

[0016] FIG. 3A-3C show that retro inverso BCL9 mimetic peptides of the invention display anti-tumor activity in an MCF7 breast cancer mouse model. Data points represent mean ±SEM BCL-26 (12.5 mg/kg) was administered to nude mice at Day 2 post-tumor inoculation (FIG. 3A; p<0.0001). BCL-87 (5 mg/kg) was administered at Day 21 post- tumor inocul ation (FIG. 3B; p<0.005). Two concentrations (1 mg/kg and 5 mg/kg) of BCL- 87 and BCL-27 were administered at Day 14 post-tumor inoculation (FIG. 3C; p£0.0004 versus control for all test peptides).

DETAILED DESCRIPTION OF THE INVENTION

[0017] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of pharmaceutics, formulation science, protein chemistry, cell biology, cell culture, molecular biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. [0018] In order that the present invention can be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention is related.

[0019] Any headings provided herein are not limitations of the various aspects or

embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0020] Ail of the references cited in this disclosure are hereby incorporated by reference in their entireties. In addition, any manufacturers’ instructions or catalogues for any products cited or mentioned herein are incorporated by reference. Documents incorporated by reference into this text, or any teachings therein, can be used in the practice of the present invention. Documents incorporated by reference into this text are not admitted to be prior art.

I. Definitions

[0021] The phraseology or terminology in this disclosure is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0022] As used in this specification and the appended claims, the singular forms“a,”

“an,” and“the” include plural referents, unless the context clearly dictates otherwise. The terms“a” (or“an”) as well as the terms“one or more” and“at least one” can be used interchangeably.

[0023] Furthermore,“and/or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term“and/or” as used in a phrase such as“A and/or B” is intended to include A and B, A or B, A (alone), and B (alone). Likewise, the term“and/or” as used in a phrase such as“A, B, and/or C” is intended to include A, B, and C; A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A (alone); B (alone); and C (alone). [0024] Wherever embodiments are described with the language“comprising,” otherwise analogous embodiments described in terms of“consisting of’ and/or“consisting essentially of’ are included.

[0025] Units, prefixes, and symbols are denoted in their Systeme International de Unites

(SI) accepted form. Numeric ranges are inclusive of the numbers defining the range, and any individual value provided herein can serve as an endpoint for a range that includes other individual values provided herein. For example, a set of values such as 1, 2, 3, 8, 9, and 10 is also a disclosure of a range of numbers from 1-10, from 1-8, from 3-9, and so forth.

Likewise, a disclosed range is a disclosure of each individual value encompassed by the range. For example, a stated range of 5-10 is also a disclosure of 5, 6, 7, 8, 9, and 10.

[0026] The terms“polypeptide,”“peptide,” and“protein” are used interchangeably

herein to refer to polymers of amino acids of any length. The polymer can be linear or branched, can comprise modified amino acids, and can be interrupted by non-amino acids. Except where indicated otherwise, e.g., for the abbreviations for the uncommon or unnatural amino acids set forth herein, the three-letter and one-letter abbreviations, as used in the art, are used herein to represent amino acid residues. Except when preceded with a“D” or in lower case, the amino acid is an L-amino acid. Groups or strings of amino acid abbreviations are used to represent peptides. Except where specifically indicated, peptides are indicated with the N-terminus of the left and the sequence is written from the N-terminus to the C- terminus.

[0027] Polypeptides, peptides, and proteins can encompass natural or synthetic

modifications, for example, disulfide bonds, lactam bridges, glycosylation, lipidation, acetylation, acylation, amidation, phosphorylation, or other manipulation or modification, such as conjugation with a labeling component or addition of a protecting group. Also included are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, amino-isobutyric acid (Aib), unnatural amino acids, such as naphthylalanine (Nal), etc.) and polypeptides comprising or consisting of D-amino acids, as well as other modifications known in the art. Polypeptides can be in one or multiple salt forms. Preferred salt forms include acetate, chloride or trifluoroacetate. In certain embodiments, the polypeptides can occur as single chains, covalent dimers, or non-covalent associated chains. Polypeptides can also be in cyclic form. Cyclic polypeptides can be prepared, for example, by bridging free amino and free carboxyl groups. Formation of the cyclic compounds can be achieved by treatment with a dehydrating agent, with suitable protection if needed. The open chain (linear form) to cyclic form reaction can involve intramolecular-cyclization. Cyclic polypeptides can also be prepared by other methods known in the art, for example, using one or more lactam bridges, hydrogen bond surrogates (Patgiri et al. 2008), hydrocarbon staples (Schafmeister et al. 2000), triazole staples (Le Chevalier Isaad et al. 2009), or disulfide bridges (Wang et al. 2006). Bridges or staples can be spaced, for example, 3, 4, 7, or 8 amino acids apart.

[0028] The term“variant” refers to a polypeptide having one or more amino acid

substitutions, deletions, and/or insertions compared to a reference sequence. Deletions and insertions can be internal and/or at one or more termini. Substitution can include the replacement of one or more amino acids with a similar or homologous amino acid(s) or a dissimilar amino acid(s). For example, some variants include alanine substitutions at one or more amino acid positions. Other substitutions include conservative substitutions that have little or no effect on the overall net charge, polarity, or hydrophobicity of the protein. Some variants include non -conservative substitutions that change the charge or polarity of the amino acid. Substitution can be with either the L- or the D-form of an amino acid.

[0029] A“retro inverse” polypeptide has a reversed amino acid sequence, relative to a native L-amino acid sequence, and is made up of D-amino acids (inverting the a-eenter chi rality of the amino acid subunits) to help maintain side-chain topology simil ar to that of the original L-amino acid peptide.

[0030] The term“conservative substitution” as used herein denotes that one or more amino acids are replaced by another, biologically similar residue. Examples include substitution of amino acid residues with similar characteristics, e.g ., small amino acids, acidic amino acids, polar amino acids, basic amino acids, hydrophobic amino acids, and aromatic amino acids. For further information concerning phenotypically silent substitutions in peptides and proteins, see, for example, Bowie et al., Science 247: 1306-1310 (1990). In the table below, conservative substitutions of amino acids are grouped by physicochemical properties; I: neutral and/or hydrophilic, II: acids and amides, III: basic, IV: hydrophobic, V: aromatic, bulky amino acids. Table

[0031] In the table below, conservative substitutions of amino acids are grouped by

physicochemical properties; VI: neutral or hydrophobic, VII: acidic, VIII: basic, IX: polar, X: aromatic.

Table

[0032] Methods of identifying conservative nucleotide and amino acid substitutions

which do not affect protein function are well-known in the art (see, e.g., Brummell et al., Biochem. 32 : 1180-1187 (1993); Kobayashi et al, Protein Eng. 12(10):879-884 (1999); and Burks et al, Proc. Natl. Acad. Sci. U.S.A. 94:412-417 (1997)).

[0033] The terms“identical” or percent“identity” in the context of two or more nucleic acids or polypeptides, refers to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary') for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms, or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences.

[0034] One such non-limiting example of a sequence alignment algorithm is described in

Karlin et al, Proc. Natl Acad Set, 87:2264-2268 (1990), as modified in Karlin et al. , Proc, Natl. Acad. Sci. , 90:5873-5877 (1993), and incorporated into the NBLAST and XBLAST programs (Altschul el ah. Nucleic Acids Res , 25:3389-3402 (1991)). In certain

embodiments, Gapped BLAST can be used as described in Altschul et al, Nucleic Acids Res. 25:3389-3402 (1997). BLAST-2, WU-BLAST-2 (Altschul et al , Methods in Enzymology, 266:460-480 (1996)), ALIGN, ALIGN-2 (Genentech, South San Francisco, California) or Megalign (DNASTAR) are additional publicly available software programs that can be used to align sequences. In certain embodiments, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (e.g. , using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 90 and a length weight of 1, 2, 3, 4, 5, or 6). In certain alternative embodiments, the GAP program in the GCG software package, which incorporates the algorithm of Needleman and Wunsch (J. Mol. Biol.

(481:444-453 (1970)), can be used to determine the percent identity between two amino acid sequences (e.g., using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5). Alternatively, in certain embodiments, the percent identity between nucleotide or amino acid sequences is determined using the algorithm of Myers and Mill er ( CABIOS 4: 11-17 (1989)). For example, the percent identity can be determined using the ALIGN program (version 2.0) and using a PAM120 with residue table, a gap length penalty of 12 and a gap penalty of 4. One skilled in the art can determine appropriate parameters for maximal alignment by particular alignment software. In certain embodiments, the default parameters of the alignment software are used. Other resources for calculating identity include methods described in Computational

Molecular Biology (Lesk ed., 1988); Biocomputing: Informatics and Genome Projects (Smith ed., 1993); Computer Analysis of Sequence Data, Part 1 (Griffin and Griffin eds , 1994); Sequence Analysis in Molecular Biology > (G. von Heinje, 1987); Sequence Analysis Primer (Gribskov et al. eds., 1991); and Cari!!o et al., SIAM J. Applied Math. , 48: 1073 (1988). [0035] A“polynucleotide,” as used herein can include one or more“nucleic acids,”

“nucleic acid molecules,” or“nucleic acid sequences,” and refers to a polymer of nucleotides of any length, and includes DNA and RNA. The polynucleotides can be

deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase. A polynucleotide can compri se modified nucleotides, such as methylated nucleotides and their analogs. The preceding description applies to all polynucleotides referred to herein, including RNA and DNA.

[0036] An“isolated” molecule is one that is in a form not found in nature, including

those which have been purified.

[0037] A“label” is a detectable compound that can be conjugated directly or indirectly to a molecule, so as to generate a“labeled” molecule. The label can be detectable on its own (e.g, radioisotope labels or fluorescent labels), or can be indirectly detected, for example, by catalyzing chemical alteration of a substrate compound or composition that is detectable (e.g., an enzymatic label) or by other means of indirect detection (e.g., biotinylation).

[0038] “Binding affinity” generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule and its binding partner (e.g., a receptor and its ligand, an antibody and its antigen, two monomers that form a dimer, etc.). Unless indicated otherwise, as used herein,“binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between members of a binding pair. The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (K _D ). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity binding partners generally bind slowly and tend to dissociate readily, whereas high-affinity binding partners generally bind faster and tend to remain bound longer.

[0039] The affinity or avidity of a molecule for its binding partner can be determined experimentally using any suitable method known in the art, e.g. , flow cytometry, enzyme- linked immunosorbent assay (ELISA), or radioimmunoassay (RIA), or kinetics (e.g., KINEXA® or BLACORE™ or OCTET® analysis). Direct binding assays as well as competitive binding assay formats can be readily employed. (See, e.g., Berzofsky et aL, “Antibody-Antigen Interactions,” In Fundamental Immunology, Paul, W. E., ed., Raven Press: New York, N.Y. (1984); Kuby, Immunology, W. H. Freeman and Company: New York, N.Y. (1992)). The measured affinity of a particular binding pair interaction can vary if measured under different conditions (e.g, salt concentration, pH, temperature). Thus, measurements of affinity and other binding parameters (e.g , K _D or Kd, K _on , K _off ) are made with standardized solution s of binding partners and a standardized buffer, as known in the art.

[0040] An“active agent” is an ingredient that is intended to furnish biological activity.

The active agent can be in association with one or more other ingredients. An active agent that is a peptide can also be referred to as an“active peptide.”

[0041] An“effective amount” of an active agent is an amount sufficient to carry out a specifically stated purpose.

[0042] The term“pharmaceutical composition” refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective and which contains no additional components that are unacceptably toxic to a subject to which the composition would be administered. Such composition can be sterile and can comprise a pharmaceutically acceptable carrier, such as physiological saline. Suitable pharmaceutical compositions can comprise one or more of a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), a stabilizing agent (e.g. polyol or amino acid), a preservative (e.g. sodium benzoate), and/or other conventional solubilizing or dispersing agents.

[0043] The terms“inhibit,”“block,” and“suppress” are used interchangeably and refer to any statistically signifi cant decrease in occurrence or activity, including full blocking of the occurrence or activity. For example,“inhibition” can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% in activity or occurrence. An“inhibitor” is a molecule, factor, or substance that produces a statistically significant decrease in the occurrence or activity of a process, pathway, or molecule.

[0044] A“neoplastic cell” or“neoplasm” typically has undergone some form of

mutation/transformation, resulting in abnormal growth as compared to normal cells or tissue of the same type. Neoplasms include morphological irregularities, as well as pathologic proliferation. Neoplastic cells can be benign or malignant. Malignant neoplasms, i.e., cancers, are distinguished from benign in that they demonstrate loss of differentiation and orientation of cells, and have the properties of invasion and metastasis. II. BCL9 Mimetic Peptides and Compositions

BCL9 Mimetic Peptides

[0045] BCL9 is a 149 kDa eukaryotic protein involved in signal transduction through the

Wnt pathway. BCL9 binds to and promotes the transcriptional activity of b-catenin. The b- catenin binding region or“HD2 domain” of BCL9 is 24-residue a-he!ix at amino acids 351- 374 of BCL9 (SEQ ID NO: 1). The full amino acid sequence of wild-type human BCL9 is set forth in NCBI Accession No. NP 004317.2.

[0046] Peptide ST-BC1 (SEQ ID NO:2) is a cyclic variant of the native BCL9 HD2

domain having a lactam bridge between residues 14 and 18. Previous studies demonstrated that an analogue of ST-BC1 having a hydrocarbon bridge between residues 14 and 18 inhibits Wnt transcriptional activity in human colon carcinoma cells and displays anti-tumor activity in mouse models (Takada et. al. 2012). The present inventors have discovered that non-conservative and linear variants of ST-BCi induce cell death in neoplastic cells and reduce tumor volume in an animal model. The discover) _' that the BCL9-derived peptides of the present invention retain their ability to specifically target and kill neoplastic cells with multiple non-conservative amino acid substitutions to the wild-type BCL9 HD2 region could not have been predicted prior to the present invention. Further a retro inverso variant was not only active, but had comparable activity relative to ST-BCI and linear“L” variants, which also could not have been predicted.

[0047] The invention provides BCL9 mimetic peptides having a modified BCL9 HD2 region and, optionally, a cell -penetrating region. BCL9 peptides of the invention are “mimetics,” meaning that they are capable of interfering with or inhibiting wild-type BCL9 activity in a cell into which they are introduced. More specifically, the BCL9 mimetic peptides of the invention are capable of binding to b-catenin and competing with native BCL9 binding to b-catenin. In some embodiments, the BCL9 mimetic peptides can downregulate expression of one or more members of the Wnt signaling pathway, for example, axin, CD44, c-Myc, cyclin Dl, LEF1, LGR5, survivin, and VEGF-A. In some embodiments, the BCL9 mimetic peptide can inhibit cell proliferation, angiogenesis, and/or cell migration. BCL9 activity can be assessed by any of several assays known in the art (Kawamoto et al. 2009; WO 2017/062518), including the cell-kill assays described herein. [0048] A“modified BCL9 HD2 region” is a sequence derived from the wild-type BCL9

HD2 region, which sequence has at least one addition, deletion, or substitution relative to the wild-type BCL9 HD2 sequence. The modified BCL9 HD2 region preferably comprises a peptide corresponding to at least positions 16-23 of SEQ ID NO: 1 and comprising at least one addition, deletion, or substitution relative to SEQ ID NO: 1. The modified BCL9 HD2 region can comprise, for example, an amino acid sequence shown in Table 1. The native BCL9 HD2 sequence (SEQ ID NO: 1) is shown as a point of reference. Substitutions in SEQ ID NO: 1 are shown in underlined bold type.

Table 1

[0049] The modified BCL9 HD2 region can be a retro inverse form and can have, for example, a D-amino acid sequence X1LX2X3QLX4X5LX6X7LA (SEQ ID NO: 142), wherein each amino acid at positions 1 -13 is independently selected from those shown in Table 2.

Table 2

[0050] In the D-amino acid HD2 domain sequences shown in Table 2, only one of

positions 4 or 8 can be alanine, i.e., if position 4 is A, position 8 is not A, and vice versa.

The BCL9 HD2 region can optionally comprise at position - l a D-amino acid or an L-amino acid selected from the group consisting of F, 1-Nal, 2-Nal, R, and W. The BCL9 IID2 region can further optionally comprise a D-amino acid or an L-amino acid selected from the group consisting of F, 1-Nal, 2-Nal, and W at position -2. In one embodiment, if position -1 is R, position 1 is F or W and/or position -2 is F, 1 -Na!, 2-Nal, or W.

[0051] Specific examples of retro inverse BCL9 HD2 regions are shown in Table 3. The retro inverse sequence of a portion of the wild-type BCL9 HD2 region (SEQ ID NO: 7) is shown as a point of reference. Substitutions in SEQ ID NO: 7 are shown in underlined bold type.

*1

[0052] The modified BCL9 HD2 region can comprise additional D-amino acids

corresponding to the full retro inverso sequence of SEQ ID NO: 1. For example, the modified BCL9 HD2 region can comprise the D-amino acid sequence

WWLARQLARLAQLARERHELQEQSL (SEQ ID NO: 121), wherein substitutions and additions relative to the retro inverso wild-type BCL9 HD2 sequence are shown in underlined bold type.

[0053] The modified BCL9 HD2 region can comprise amino acids of mixed chirality, such that one or more amino acids in the peptide are in the L form and one or more amino acids are in the D form. For example, an L-peptide can comprise one or more D-amino acids. Likewise, a retro inverso D-peptide can comprise one or more L-amino acids. In certain embodiments, the BCL9 HD2 region has a sequence selected from the group consisting of:

(i) F _D R _L [WLARQLARLAQLA] _D (SEQ ID NO: 103);

wherein substitutions and additions relative to the retro inverse wild-type BCL9 HD2 sequence (SEQ ID NO: 7) are shown in underlined hold type

[0054] Variants of these sequences are also included in the scope of the invention. BCL9 mimetic peptides of the invention can have an HD2 region of at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to those sequences disclosed herein.

[0055] In embodiments wherein the BCL9 mimetic peptide comprises another active peptide, such as a cell-penetrating region or an RGD-like sequence, the active peptide is operably linked to the modified BCL9 HD2 region. In some embodiments, the active peptide is covalently linked to the modified BCL9 HD2 region, for example, via a peptide bond, a disulfide bond, a thioether bond, or a linker known in the art. Exemplary linkers include, but are not limited to, a substituted alkyl, a substituted cycloalkyl, polyethylene glycol, and derivatives thereof. Linkers can be cleavable after the peptide is delivered into a cell. An active peptide and a modified BCL9 HD2 region linked directly by an amide bond may be referred to as a“fusion” Fusions can contain an amino acid linker sequence between the active peptide and the modified BCL9 HD2 region, as discussed above with respect to active peptides. The active peptide can be linked to the N-terminus or the C -terminus of the modified BCL9 HD2 region, or via a residue side chain. The active peptide and modified BCL9 TID2 region can have the same or opposite chirality.

[0056] Cell-penetrating BCL9 mimetic peptides of the invention can comprise any

combination of cell-penetrating and modified BCL9 HD2 regions disclosed herein. Non limiting examples of such peptides are shown in Table 4. The ceil -penetrating region is italicized. Peptide BCL-21 comprises the native BCL9 HD2 sequence and is inefficient at inhibiting cell proliferation. Substitutions relative to the native BCL9 HD2 sequence are shown in underlined bold type. Sapience.004. WOl

PATENT

[0057] Retro inverse forms of BCL9 mimetic peptides are also included. Exemplary embodiments of cell-penetrating retro inverse BCL9 mimetic peptides are shown in Table 5.

Table 5

[0058] The cell-penetrating and RGD-like regions are italicized. Substitutions and

additions relative to the retro inverso wild-type BCL9 HD2 sequence (SEQ ID NO: 7) are shown in underlined bold type. The invention also includes peptides comprising the BCL9 HD2 regions shown in Table 5 and a different active peptide, such as a different cell- penetrating region, and peptides comprising the BCL9-HD2 regions shown in Table 5 without an active peptide.

[0059] BCL9 mimetic peptides of the invention can comprise amino acids of mixed chirality, such that one or more amino acids in the peptide are in the L form and one or more amino acids are in the D form. Non-limiting examples of BCL9 mimetic peptides having mixed chirality are shown in Table 6.

Table 6

[0060] D and L subscripts denote chirality of the amino acids. The cell-penetrating region is italicized. Substitutions and additions relative to the retro inverse wild-type BCL9 HD2 sequence (SEQ ID NO: 7) are shown in underlined bold type. The invention also includes peptides comprising the BCL9 HD2 regions shown in Table 6 and a different active peptide, such as a different cell-penetrating region, and peptides comprising the BCL9-HD2 regions shown in Table 6 without an active peptide.

[0061] BCL9 mimetic peptides of the invention include peptides having at least about

80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to those sequences disclosed herein. [0062] BCL9 mimetic peptides of the invention are preferably 7, 8, 9, 10, 11, 12, 13, 14,

15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length, including ranges having any of those lengths as endpoints, for example, 13-35 amino acids.

[0063] The BCL9 mimetic peptides can have a modified N-terminus and/or a modified

C-terminus. For example, BCL9 mimetic peptides can optionally include an N-terminal acetyl group and/or a C-terminal amide group. Other examples of optional N-terminal and/or C-terminal groups include hydrophobic groups, such as a linear or cyclic C2-C18 aliphatic or aromatic hydrocarbon, a naphthyl group, a phenyl group, an octanoyl group, and a valeryl group, including an isovalery! group. In some embodiments, the BCL9 mimetic peptide comprises a linker or spacer between the peptide and the hydrophobic group. Such linkers or spacers include, for example, aminohexanoic acid, beta-alanine, substituted alkyls, substituted cycloalkyls, and polyethylene glycol.

[0064] BCL9 mimeti c peptides of the inventi on can optionally be cyclic. For example,

BCL9 mimetic peptides of the invention can include one or more lactam bridges. A lactam bridge is preferably, but not necessarily, created between side chains spaced three, four, seven, or eight amino acid residues apart (i.e., BxxB, BxxxB, BxxxxxxB, BxxxxxxxB). Lactam bridges can be formed, for example, between the side chains of Asp or GIu and Lys or Grn. Amino acid substitutions can be made at the site of the lactam bri dge to facilitate the linkage.

[0065] BCL9 mimetic peptides of the invention can optionally include one or more

epitope and/or affinity tags, such as for purification or detection. Non-limiting examples of such tags include FLAG, HA, His, Myc, GST, and the like. BCL9 mimetic peptides of the invention can optionally include one or more labels.

[0066] In certain aspects, the invention provides a composition, e.g., a pharmaceutical composition, comprising a BCL9 mimetic peptide of the invention, optionally further comprising one or more carriers, diluents, excipients, or other additives.

[0067] Also within the scope of the invention are kits compri sing the BCL9 mimetic peptides and compositions as provided herein and, optionally, instaictions for use. The kit can further contain at least one additional reagent, and/or one or more additional active agent.

Kits typically include a label indicating the intended use of the contents of the kit. In this context, the term“label” includes any writing or recorded material supplied on or with the kit, or that otherwise accompanies the kit.

[0068] The BCL9 mimetic peptides of the invention can be used to inhibit proliferation of and/or to promote cytotoxicity in a neoplastic cell. Proliferation and cytotoxicity can be measured by known assays, including the cell kill assays described herein.

Cell Targeting

[0069] BCL9 mimetic peptides of the invention can be introduced into target cells by methods known in the art. The method of introduction chosen will depend, for example, on the intended application.

[0070] In some instances, DNA or RNA encoding the BCL9 mimetic peptide can be delivered to and expressed in a target cell. Delivery can be accomplished via any suitable vector, depending on the application. Examples of vectors include plasmid, cosmid, phage, bacterial, yeast, and viral vectors prepared, for example, from retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses, and envelope-pseudotyped viruses. Vectors can be introduced into cells, for example, using nanoparticles, hydrodynamic delivery, electroporation, sonoporation, calcium phosphate precipitation, or cationic polymers such as DEAE-dextran. Vectors can be complexed with lipids, such as

encapsulated in liposomes, or associated with cationic condensing agents.

[0071] BCL9 mimetic peptides of the invention can be delivered to cells via mechanisms that exploit cellular receptors. Examples of such mechanisms include antibody-drug conjugates, chimeric antigen receptors, multiple antigen presentation (MAP) systems, and integrin-targeting, RGD-like sequences. Examples of RGD-like sequences include GRGDS (SEQ ID NO: 28) and GRGDNP (SEQ ID NO: 29). BCL9 mimetic peptides of the invention can comprise one or more RGD-like sequences, such as two, three, four, or five RGD-like sequences, linked as described herein or by any method known in the art . The one or more RGD-like sequence(s) can be incorporated to the N-terminal or C-terminal side of the BCL9 HD2 region. Such RGD-like sequences can also be in retro inverso form, independently of one another and of the BCL9 HD2 region. One particular example of a retro inverso RGD- like sequence is PSDGRG (SEQ ID NO: 75). Alternatively, BCL9 mimetic peptides can be encapsulated and delivered to ceils in vesicles, such as exosomes or liposomes, or in micelles. Another method for introducing BCL9 mimetic peptides into ceils is via cyclization, for example, using hydrocarbon staples (Bernal et al. 2007; Bird et al. 2016) or other cyclization methods known in the art.

[0072] Certain BCL9 mimetic peptides of the present invention comprise a cell- penetrating domain or cell -penetrating peptide (CPP). The terms“cell-penetrating domain,” “cell-penetrating region,” and“cell-penetrating peptide” are used interchangeably herein.

[0073] CPPs are short (typically about 6-40 amino acids) peptides that are able to cross cell membranes. Many CPPs are capable of crossing the blood-brain barrier (BBB). in some embodiments, the CPP is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 amino acids in length, including ranges having any of those lengths as endpoints, for example, 10-30 amino acids. CPPs have the ability to transport covalently or non-covalent!y linked molecular cargo, such as polypeptides, polynucleotides, and nanoparticles, across cell membranes and the BBB. The translocation can be endocytotic or energy-independent (i.e., non-endocytotic) via translocation. Numerous CPPs are described and characterized in the literature (see, e.g., Handbook of Cell-Penetrating Peptides (2d ed. Ulo Langei ed., 2007); Herve et al. 2008; Heitz et al. 2009; Munyendo et al. 2012; Zou et al. 2013; Krautwald et al. 2016). A curated database of CPPs is maintained at crdd.osdd.net/raghava/cppsite (Gautam et al. 2012).

[0074] Peptides referred to as nuclear localization sequences (NLSs) are a subset of

CPPs. The classical NLS contains one (monopartite) or two (bipartite) regions of basic amino acids. Consensus sequences of cl assical monopartite and bipartite NLSs are, respectively, K(K/R)X(K/R) (SEQ ID NO: 30) and (K/R)(K/R)Xio.i2(K/R) ₃ /5 (SEQ ID NO: 31), where 3/5 indicates that at least 3 of 5 consecutive amino acids are lysine or arginine (Kosugi et al. 2009). An NLS sequence from SV40 large T antigen, PKKKRKV (SEQ ID NO: 57), is an example of a classical monopartite NLS, while an NLS sequence from nucleop!asmin, KRPA.ATKKAGQAKKK (SEQ ID NO: 44) is an example of a classical bipartite NLS (Lange et al. 2007; Kosugi et al. 2009). There are also numerous non-classical NLSs, such as those from ribonucleoproteins (RNPs) hnRNP Al, hnRNP K, and U snRNP (Mattaj et al 1998).

[0075] Non-limiting examples of CPPs suitable for use in the present invention include peptides derived from proteins, such as from Drosophila antennapedia transcription factor (Penetratin and its derivatives RL-16 and EB1) (Derossi et al 1998; Thoren et al. 2000; Lundberg et al 2007; Alves et al. 2008); from HIV-1 trans-activator of transcription (Tat) (Vives et al 1997; Hallbrink et al 2001); from rabies vims glycoprotein (RVG) (Kumar et al. 2007); from herpes simplex vims VP22 (Elliott et al. 1997); from antimicrobial protegrin 1 (SynB) (Rousselle et al. 2001), from rat insulin 1 gene enhancer protein (pISl) (Kilk et al 2001; Magzoub et al 2001); from murine vascular endothelial cadherein (pVEC) (Elmquist et al. 2001); from human calcitonin (hCT) (Schmidt et al 1998); and from fibroblast growth factor 4 (FGF4) (Jo et al 2005 ) CPPs suitable for use in the invention also include synthetic and chimeric peptides, such as Transportan (TP) and its derivatives (Pooga et al 1998; Soomets et al 2000); membrane translocating sequences (MTSs) (Brodsky et al 1998; Lindgren et al 2000; Zhao et al 2001), such as the MPS peptide (also known as fusion sequence-based peptide or FBP) (Chaloin et al. 1998); sequence signal -based peptide (SBP) (Chaloin et al 1997); model amphipathic peptide (MAP) (Oehlke et al 1998; Scheller et al 1999; Hallbrink et al 2001), translocating peptide 2 (TP2) (Cruz et al 2013), MPG (Morris et al 1997; Kwon et al. 2009), Pep-1 (Morris et al 2001 ; Munoz-Morris et al. 2007), and poly-arginine (e.g., R ₇ -R ₁₂ (SEQ ID NO: 144)) (Mitchell et al 2000; Wender et al 2000; Futaki et al 2001; Suzuki et al 2002). Representative but non-limiting sequences are shown in Table 7.

Table 7

[0076] Because the function of CPPs depends on their physical characteristics rather than sequence-specific interactions, they can have the reverse sequence and/or reverse chirality as those provided in Table 7 and/or known in the art. For example, retro inverse forms of the CPPs (reverse sequence and reverse chirality) are suitable for use in the invention. Examples of retro inverso CPPs include those having the D-amino acid sequence

KKWKMRRN QF WIK IQR (SEQ ID NO: 71), KKWKMRRNQFWLKLQR (SEQ ID NO: 72), RRRQRRKKRGY (SEQ ID NO: 73), KLTPV (SEQ ID NO: 74), or OLTPV (SEQ ID NO: 143). Variants of these sequences with one or more amino acid additions, deletions, and/or substitutions that retain the ability to cross cell membranes and/or the BBB are also suitable for use in the invention. The BCL9 mimetic peptides of the invention can include a cell-penetrating domain having at least about 60%, 61%, 62%, 63%, 64%,, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% _> , 89%, 90%, 91%, 92%, 93%, 94%, 95%,, 96%, 97%, 98%, or 99% identity to the exemplar} _' sequences provided in Table 7. The effect of the amino acid addition(s), deletion(s), and/or substitution(s) on the ability of the CPP to mediate cell penetration can be tested using methods known in the art.

III. Methods of Preparation

[0077] BCL9 mimetic peptides of the invention can be chemically synthesized, for

example, using solid-phase peptide synthesis or solution-method peptide synthesis, or can be expressed using recombinant methods. Synthesis or expression may occur as fragments of the peptide which are subsequently combined either chemically or enzymatically.

[0078] Accordingly, also provided are nucleic acid molecules encoding BCL9 mimetic peptides of the invention. Such nucleic acids can be constructed by chemical synthesis using an oligonucleotide synthesizer. Nucleic acid molecules of the invention can be designed based on the amino acid sequence of the desired BCL9 mimetic peptide and selection of those codons that are favored in the host cell in which the recombinant BCL9 mimetic peptide will be produced. Standard methods can be applied to synthesize a nucleic acid molecule encoding an BCL9 mimetic peptide of interest.

[0079] Once prepared, the nucleic acid encoding a particular BCL9 mimetic peptide can be inserted into an expression vector and operably linked to an expression control sequence appropriate for expression of the peptide in a desired host. In order to obtain high expression levels of the BCL9 mimetic peptide, the nucleic acid can be operably linked to or associated with transcriptional and translational expression control sequences that are functional in the chosen expression host.

[0080] A wide variety of expression host/vector combinations can be employed to

anyone known in the art. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV4G, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli , including pCRl, pBR322, pMB9 and their derivatives, wider host range plasmids, such as M13, and filamentous single- stranded DNA phages.

[0081] Suitable host cells include prokaryotes, yeast, insect, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram negative or gram positive organisms, for example E. coli or bacilli. Higher eukaryotic cells can be established or cell lines of mammalian origin, examples of which include Pichia pastoris, 293 cells, COS-7 cells, L cells, C127 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, HeLa cells, and BHK cells. Cell-free translation systems can also be employed.

EXAMPLES

[0082] Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples. It wall be apparent to those skilled in the art that many modifications, both to materials and methods, can be practiced without departing from the scope of the present discl osure.

Example 1. BCL9 Peptides Display Anti-Proliferative Activity in Cancer Cells In Vitro

[0083] We generated a panel of peptides containing a cell-penetrating region and a BCL9

HD2 domain and compared their activity to a previously described cyclic peptide (Takada et al 2012), ST-BC1. The BCL9 peptides discussed in this Example are summarized in Table 8

Table 8

[0084] ST-BC1 contains a lactam bridge between the bolded, underlined residues. The other five peptides are linear, with a TAT cell-penetrating region, shown in italics. Peptide BCL-21 contains the native HD2 domain, while each of peptides BCL-22-25 has amino acid substitutions in the native BCL9 HD2 domain. In addition to substitutions, peptide BCL-25 also has a shortened HD2 domain relative to the native sequence.

[0085] MCF-7 breast cancer cells set at a density of 2.5 x 10 ⁴ cells/well in 150 pL of

MEM medium + 10% fetal bovine serum (FBS) in a 96 well dish. Lyophilized BCL9 peptides were reconstituted at a concentration of 10 mg/mL in 270 mM trehalose buffer and added to each well at a volume of 50 pL to a final concentration range of 2.5-40 mM. Cells were incubated with BCL9 peptides for 96 hours at 37°C.

[0086] Cell viability was quantified by spectrophotometry using Roche MTT cell

proliferation assay kit according to manufacturer’s instructions. Briefly, cells were washed with PBS, and incubated in fresh MEM medium with 10% FBS, 1% penicillin/streptomycin, and 1% non-essential amino acids, plus 10 pL MTT reagent at 37°C for 4 hours. After 4 hours, 100 pL of solubilization solution was added, and cells were incubated overnight at 37°C and 5% carbon dioxide. Absorbance was measured at OD570 nm with a reference of OD650 nm. The degree of absorption is proportional to the number of living cells.

Percentage of absorbance relative to untreated controls was quantified and presented as % Cell Viability. [0087] Modified BCL9 peptides of the invention, but not BCL-21 having the native BCL9 HD2 domain sequence, demonstrated equal or greater anti-proliferative activity in MCF7 breast cancer cells compared to peptide ST-BC1 (EC ₅₀ values <10 µM) (FIG.1). In functional assays, EC ₅₀ is the concentration that reduces a biological response by 50% of its maximum. In the case of BLC9 peptides, EC ₅₀ is measured as the concentration that reduces cell viability by 50% of its maximum. EC ₅₀ can be calculated by any number of means known in the art.

Example 2. Retro Inverso BCL9 Peptides Display Anti-Proliferative Activity in Cancer

Cells In Vitro

[0088] We prepared retro-inverso peptides: BCL-26, with the D-amino acid sequence FLMRQLDRLTQLAKLTPV (SEQ ID NO: 26), and BCL-27, with the D-amino acid sequence WWLARQLARLAQLAKLTPV (SEQ ID NO: 27). A cell-penetrating region derived from Bax-inhibiting peptide is shown in italics. In BCL-26, the sequence of the HD2 domain is shortened by 11 amino acids and substituted at 2 positions relative to the native sequence. In BCL-27, the sequence of the HD2 domain is shortened by 11 amino acids, substituted at 6 positions, and contains an additional N-terminal tryptophan relative to the native sequence.

[0089] We examined the cytotoxicity of peptides BCL-26 and BCL-27 using an assay in HL60 promyelocytic leukemia cells. HL60 PML suspension cells were set at a density of 3.5 x 10 ³ cells/well in 150 µL of RPMI + 1.5% fetal bovine serum (FBS) in a 96 well dish. BCL- 26 or BCL-27, reconstituted at a concentration of 10 mg/mL in 20 mM His, pH 7.5, was added to each well at a volume of 50 µL to a final concentration range of 0-80 µM. Cells were incubated with peptide for 48 hours at 37°C. Cell viability was quantified by flow cytometry using abcam Annexin V FITC apoptosis detection kit. Briefly, cells were washed with PBS and resuspended in 1x assay buffer containing Annexin V FITC and propidium iodide (PI). Annexin V detects apoptotic cells, and PI stains dead cells. After staining, apoptotic cells show green fluorescence, dead cells show red and green fluorescence, and live cells show little or no fluorescence. Cells were selected for analysis based on forward scatter (FSC) vs. side scatter (SSC), and analyzed by BD Accuri C6 Plus flow cytometer to detect Annexin V-FITC binding (Ex = 488 nm; Em = 530 nm) using FITC signal detector and PI staining by the phycoerythrin emission signal detector. Percentage of Annexin V ^Iow and PI ^low were quantified and presented as % Viability. The retro inverse peptides had comparable activity to the standard peptides tested in Example 1 (FIG. 2A-2B).

[0090] We examined the cytotoxicity of additional retro inverse and mixed chirality

BCL9 mimetic peptides in HL60 cells using the assay described above. Results are shown in Table 9.

Table 9

+++ indicates EC50 < 2 mM; ++ indicates EC50 = 2-10 mM; + indicates EC50 > 10 mM; NA indicates Not Active

[0091] D and L subscripts denote chirality of the amino acids. Substitutions and

additions relative to the retro inverse wild-type BCL9 HD2 sequence (SEQ ID NO: 7) are shown in underlined bold type. The cell-penetrating and RGD-like regions are italicized. As expected, BCE-12 displayed no cytotoxic activity because it lacks a sequence for cell penetration.

[0092] Lack of cytotoxic activity by BCL-134 demonstrates the requirement for a

positively charged amino acid in at least one of positions 4 or 8 of the HD2 domain, relative to SEQ ID NO: 7. (Compare the activity of BCL-133 with that of BCL-134.) [0093] Lack of cytotoxic activity by BCL-138 demonstrates the requirement that at least one of the two N-terminai amino acids of the HD2 domain is hydrophobic. (Compare the activity of BCL-91b with that of BCL-138.)

Example 3. Retro Inverso BCL9 Peptide Displays Anti-Tumor Activity In Vivo

[0094] In this experiment, we examined the effect of peptide BCL-26 on tumor volume in an MCF7 subcutaneous tumor model. Briefly, 2 x 10 ⁶ MCF7 breast cancer cells, suspended 1 : 1 in Matrigel, were implanted via subcutaneous injection into the axilla of NU/J mice. Peptide BCL-26 was administered at a dose of 12.5 mg/kg via subcutaneous injection three times weekly for three weeks. Dosing was initiated on day 2 post tumor inoculation, with an average starting tumor volume of about 25 mm ³ . Peptide BCL-26 significantly reduced tumor volume relative to vehicle (FIG. 3 A).

[0095] We also examined the effect of BCL9 mimetic peptides in established tumors.

MCF7 cells were implanted into mice as described above. Peptide BCL-87 was administered at a dose of 5 mg/kg via subcutaneous injection three times weekly for three weeks. Dosing was initiated on day 21 post tumor inoculation, with an average starting tumor volume of about 470 mm ³ . Peptide BCL-87 significantly reduced tumor volume relative to vehicle (FIG. 3B).

[0096] We additionally examined the effect of different concentrations of mimetic

peptides in established tumors. MCF7 cells were implanted into mice as described above. Peptides BCL-27 and BCL-87 were administered at a dose of 1 mg/kg or 5 mg/kg via subcutaneous injection three times weekly for three weeks. Dosing was initiated on day 14 post tumor inoculation, with an average starting tumor volume of about 340 mm ³ . Both peptides significantly reduced tumor volume relative to vehicle and to a peptide control, BCL-134 (FIG. 3C).

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The present invention is further described by the following claims.