Beta-catenin depot detection and modulation assays

Abstract

The present application provides, in some aspects, methods of determining a beta-catenin depot and uses thereof, for example, to identify compounds that modulate, such as increase, a beta-catenin depot in a cell. Also provided herein, in other aspects, are related methods, such as methods of identifying compounds for the treatment of beta-catenin associated diseases, kits, and useful compounds identified thereby.

Description

Cross-reference to related applications

[0001] This application claims priority and interest in U.S. Provisional Patent Application No. 63 / 512,560, filed July 7, 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Reference to the electronic sequence list The contents of the electronic sequence list (185992001940seqlist.xml; size: 2,666 bytes; and creation date: July 1, 2024) are incorporated herein by reference in their entirety. Technical Field

[0003] This application relates to the field of biological condensates. Background Technology

[0004] Conventional applications of disease research and therapeutic drug discovery focus on identifying single biomolecules (molecular targets) that cause or mediate disease-related biology, such as via known cellular pathways. This “single-target” approach to disease research and therapeutic drug discovery has significant limitations because in vivo systems are extremely complex, and our understanding of cellular pathways and the actions and interactions of biomolecules is often incomplete. Furthermore, not all individual biomolecules are suitable drug targets from a standalone perspective. These challenges result in the current lack of novel therapeutics to address unmet medical needs. In the search for novel therapeutics, bioaggregates are currently being evaluated as targets and / or tools for drug discovery. Some preliminary studies in the field of bioaggregates have observed chemical entities capable of indiscriminately dissolving bioaggregates and / or preventing their formation. However, the influence of such broadly affected chemical entities can lead to undesirable drug toxicity and off-target properties. Therefore, there remains a need to identify novel bioaggregates for therapeutic purposes, as well as new methods for identifying therapeutic candidates that selectively modulate such aggregates and / or their components. Summary of the Invention

[0005] In some aspects, a method is provided for identifying compounds capable of regulating intracellular β-catenin reservoirs, the method comprising: (a) contacting a composition containing cells with the compound; (b) determining the β-catenin reservoir of the composition containing cells or a derivative thereof; and (c) identifying compounds regulating β-catenin reservoirs in cells by comparing the β-catenin reservoirs determined in cells with a reference.

[0006] In some embodiments, comparing a β-catenin reservoir measured in cells with a reference is based on one or more of the following properties of the β-catenin reservoir: (i) the presence of the β-catenin reservoir; (ii) the number of β-catenin reservoirs; (iii) the size of the β-catenin reservoir; (iv) the surface area of ​​the β-catenin reservoir; (v) the cellular localization of the β-catenin reservoir; (vi) the distribution of the β-catenin reservoir; (vii) the ratio of the amount of the β-catenin reservoir to the amount of the reference aggregate; or (viii) the composition of the β-catenin reservoir. In some embodiments, regulation of the β-catenin reservoir is based on an increase in the number of β-catenin reservoirs in cells compared to a reference. In some embodiments, regulation of the β-catenin reservoir in cells is based on an increase in the number of β-catenin reservoirs in the cell nucleus compared to a reference. In some embodiments, the regulation of cellular β-catenin reservoirs is based on an increase in the presence of cellular β-catenin reservoirs compared to a reference. In some embodiments, the regulation of cellular β-catenin reservoirs is based on an increase in the presence of β-catenin reservoirs in the cell nucleus compared to a reference.

[0007] In some embodiments, the assay of a β-catenin reservoir includes detecting an inactive form of the β-catenin peptide. In some embodiments, detecting the inactive form of the β-catenin peptide includes contacting a cell-containing composition or a derivative of that composition with a detectable reagent that specifically recognizes the inactive form of the β-catenin peptide. In some embodiments, the detectable reagent is an antibody that specifically recognizes the inactive form of the β-catenin peptide. In some embodiments, the assay includes detecting the detectable reagent that specifically recognizes the inactive form of the β-catenin peptide. In some embodiments, the assay includes imaging at least a portion of a cell. In some embodiments, the detectable reagent that specifically recognizes the inactive form of the β-catenin peptide is a fluorescently labeled antibody that specifically recognizes the inactive form of the β-catenin peptide. In some embodiments, the detectable reagent that specifically recognizes the inactive form of the β-catenin peptide is detected using a fluorescently labeled secondary antibody that specifically recognizes the detectable reagent. In some embodiments, the detectable reagent that specifically recognizes the inactive form of the β-catenin polypeptide specifically recognizes phosphorylation at one or more of S33, S37, T41, or S45 of the inactive form of the β-catenin polypeptide, wherein the amino acid position is relative to the amino acid sequence of SEQ ID NO:1. In some embodiments, the detectable reagent that specifically recognizes the inactive form of the β-catenin polypeptide specifically recognizes phosphorylation at T41 and / or S45. In some embodiments, the detectable reagent that specifically recognizes the inactive form of the β-catenin polypeptide specifically recognizes phosphorylation at both T41 and S45. In some embodiments, the detectable reagent that specifically recognizes the β-catenin polypeptide is a 23H16L13 antibody clone.

[0008] In some embodiments, comparing a β-catenin reservoir measured in cells with a reference is based on one or more of the following properties of the inactive form of the β-catenin polypeptide associated with the β-catenin reservoir: (i) the localization of the inactive form of the β-catenin polypeptide; (ii) the amount of the inactive form of the β-catenin polypeptide; (iii) the distribution of the inactive form of the β-catenin polypeptide; (iv) the aggregate distribution of the inactive form of the β-catenin polypeptide in the β-catenin reservoir and / or one or more other aggregates; (v) the functional activity associated with the inactive form of the β-catenin polypeptide; (vi) the aggregation of the inactive form of the β-catenin polypeptide; (vii) the post-translational modification state of the inactive form of the β-catenin polypeptide; or (viii) the amount of degradation products of the inactive form of the β-catenin polypeptide.

[0009] In some implementations, the regulation (e.g., increase) of the β-catenin reservoir in the cell is based on an increase in the amount of inactive form of β-catenin polypeptide in the β-catenin reservoir compared to a reference.

[0010] In some implementations, the determination of the β-catenin reservoir includes the detection of phosphorylated H2A.X (P-2A.X) and / or DNA damage checkpoint mediator 1 (MDC1).

[0011] In some embodiments, the cells are models of abnormal Wnt / β-catenin signaling. In some embodiments, the cells are models of proliferative pathology. In some embodiments, the cells are models of cancer. In some embodiments, the cells are cancer cells. In some embodiments, the cancer cells are human osteosarcoma U2OS cells or human colon adenocarcinoma DLD-1 cells.

[0012] In some implementations, the cells are models of autosomal dominant polycystic kidney disease (ADPKD), hereditary familial adenomatous polyposis (FAP), Alzheimer's disease, or metabolic diseases such as obesity and / or insulin resistance.

[0013] In some embodiments, the inactive form of the β-catenin polypeptide is a wild-type β-catenin polypeptide. In some embodiments, the inactive form of the β-catenin polypeptide is a mutant β-catenin polypeptide.

[0014] In some embodiments, the assay further includes contacting a composition comprising the cells or a derivative thereof with a second detectable reagent that specifically recognizes the active form of the β-catenin polypeptide containing phosphorylated amino acids at S552 and / or S675, wherein the amino acid positions are relative to the amino acid sequence of SEQ ID NO:1.

[0015] In some embodiments, the method further includes contacting the cell-containing composition or a derivative thereof with a fixative prior to the determination in step (b).

[0016] In some implementations, the method also includes determining one or more cellular characteristics of the cells.

[0017] In some embodiments, references are based on: (i) a composition containing cells not in contact with the compound; (ii) a composition containing cells mixed with a control medium and not in contact with the compound; (iii) a desired β-catenin profile; (iv) a reference condensate or reference peptide within the cells; and / or (v) a reference composition containing cells.

[0018] In some embodiments, the method further includes subjecting the composition containing the cells to conditions that promote the formation of a β-catenin reservoir before contacting the composition containing the cells with the compound.

[0019] In some embodiments, the method further includes using a second cell-based assay and / or in vitro assay to evaluate the identified compound.

[0020] In some other aspects, this document provides a method for identifying β-catenin reservoirs in cells, the method comprising: (a) contacting a composition containing cells with a detectable reagent that specifically recognizes an inactive form of β-catenin polypeptide phosphorylated at T41 and / or S45; and (b) detecting the detectable reagent, wherein detecting the detectable reagent intracellularly (e.g., in aggregates) identifies β-catenin reservoirs in cells. In some embodiments, the detectable reagent is an antibody that specifically recognizes an inactive form of β-catenin polypeptide phosphorylated at T41 and / or S45. In some embodiments, the detectable reagent is an antibody that specifically recognizes an inactive form of β-catenin polypeptide phosphorylated at T41 and S45. In some embodiments, the detection includes imaging at least a portion of the cell. In some embodiments, the detectable reagent is a fluorescently labeled antibody that specifically recognizes an inactive form of β-catenin polypeptide phosphorylated at T41 and / or S45. In some embodiments, the detectable reagent is detected using a fluorescently labeled second antibody that specifically recognizes the detectable reagent. In some embodiments, the method further includes contacting the cell-containing composition with a fixative before contacting the detectable reagent. In some embodiments, the method further includes measuring the signal of the detectable reagent. In some embodiments, the method further includes comparing the signal with a reference. In some embodiments, the reference is based on: (i) a composition containing cells not contacted with the detectable reagent; (ii) a composition containing cells mixed with a control medium and not contacted with the detectable reagent; (iii) a reference signal; (iv) a reference condensate or reference peptide within the cells; and / or (v) a reference composition containing cells.

[0021] In some other aspects, this document provides methods for identifying compounds for treating β-catenin-related diseases, wherein the methods include identifying the compounds according to the methods described herein. In some embodiments, β-catenin-related diseases are selected from the group consisting of cancer, autosomal dominant polycystic kidney disease (ADPKD), hereditary familial adenomatous polyposis (FAP), Alzheimer's disease, or metabolic diseases.

[0022] In some other respects, this article provides compounds identified by the methods described herein.

[0023] In some other respects, this document provides a method for identifying a reservoir of β-catenin in cells, the method comprising: (a) contacting a composition containing cells with a first detectable reagent that specifically recognizes a β-catenin polypeptide; (b) contacting the composition containing cells with a second detectable reagent that specifically recognizes phosphorylated H2A.X (P-2A.X) or DNA damage checkpoint mediator 1 (MDC1); and (c) detecting the first and second detectable reagents; wherein the detection of the colocalization of the first and second detectable reagents within the cells identifies a reservoir of β-catenin in the cells. Attached Figure Description

[0024] Figure 1 Images are shown of cells expressing Halo-β-catenin or labeled with antibodies that specifically bind to either inactive or active β-catenin (“ABC”). Antibodies that specifically bind to inactive β-catenin allow visualization of the β-catenin reservoir, while antibodies that specifically bind to active β-catenin do not due to high background noise. DAPI was used for staining the cell nuclei.

[0025] Figure 2 The dose-response curves for compound A in wt U2OS cells are shown.

[0026] Figure 3 Immunofluorescence (IF) images of healthy and malignant colon cells after treatment with a small molecule aggregate regulator B (compound B), which was identified and optimized from a screening of 20,000 compounds, are shown. C-mod treatment of malignant cells resulted in the segregation of β-catenin into aggregates.

[0027] Figure 4 The study showed that treatment with compound B, identified and optimized from a screening of 20,000 compounds, resulted in more selective killing of cancerous colon cells than of healthy colon cells.

[0028] Figure 5 Gene set enrichment analysis (GSEA) plots are shown, revealing the selective downregulation of Wnt / β-catenin and LEF1 target genes in colorectal cancer cells when treated with compound B, which is a β-catenin reservoir inducer.

[0029] Figure 6 The results of treatment with compound B on 99 cancer cell lines are shown. These results indicate that compound B is active in a wide range of Wnt-related cancers.

[0030] Figure 7 Compound B was shown to cause tumor growth arrest in a patient-derived xenograft (PDX) model derived from heavily pretreated CRC patients with high β-catenin expression. FOLFOX is a combination of chemotherapy drugs representing the standard of care for patients with metastatic colorectal cancer. QW = once weekly; BID = twice daily; QD = once daily. Detailed Implementation

[0031] In some aspects, this application provides methods for identifying compounds that regulate β-catenin reservoirs in cells. This disclosure is based, at least in part, on the inventors’ ability to identify and measure specific β-catenin reservoirs, which are aggregates containing certain inactive forms of β-catenin, and can be used to identify compounds that produce desired results in cells exhibiting aberrant Wnt / β-catenin signaling, which leads to growth-related pathologies such as cancer and other diseases. For example, the ability to measure β-catenin reservoirs using imaging techniques enables the use of high-throughput compound screening methods to identify compounds that regulate aberrant Wnt / β-catenin signaling. As described herein, the identification of β-catenin reservoirs allows for the discovery of co-localized macromolecules, which further expands the range of macromolecules that can be used to evaluate β-catenin reservoirs. Furthermore, various mutations in the Wnt / β-catenin signaling pathway have been found to be associated with cancer and lead to increased β-catenin signaling. However, in a group of cell lines, regardless of the mutation (including β-catenin hotspot mutations), a single phenotype was found to be associated with the identification of therapeutically useful compounds: β-catenin reservoir formation. The findings described in this paper regarding the specific measurement of β-catenin reservoirs represent a significant advance in the fields of condensate and drug discovery methodologies, especially considering that, for example, some methods cannot measure such β-catenin reservoirs.

[0032] Therefore, in some aspects, this document provides a method for identifying compounds capable of regulating intracellular β-catenin reservoirs, the method comprising: (a) contacting a cell-containing composition with the compound; (b) determining the β-catenin reservoir of the cell-containing composition or a derivative thereof; and (c) identifying compounds regulating cellular β-catenin reservoirs by comparing the β-catenin reservoirs determined in the cells with a reference.

[0033] In other respects, this document provides a method for identifying β-catenin reservoirs in cells, the method comprising: (a) contacting a cell-containing composition with a detectable reagent that specifically recognizes an inactive form of β-catenin polypeptide phosphorylated at T41 and / or S45; and (b) detecting the detectable reagent, wherein detecting the detectable reagent intracellularly (e.g., in aggregates) identifies β-catenin reservoirs in cells. In some aspects described herein, the detectable reagent that specifically recognizes an inactive form of β-catenin polypeptide phosphorylated at T41 and / or S45 is a detectable reagent that determines the presence of a target in the presence of a heterologous molecular population. In some embodiments, the detectable reagent may also bind to other molecules, including biomolecules, to a degree of affinity and / or affinity.

[0034] In other respects, this document provides methods for identifying compounds for treating β-catenin-related diseases, wherein the methods include identifying the compounds according to the methods described herein. In some embodiments, β-catenin-related diseases are selected from the group consisting of cancer, autosomal dominant polycystic kidney disease (ADPKD), hereditary familial adenomatous polyposis (FAP), Alzheimer's disease, or metabolic diseases.

[0035] In some other respects, this article provides compounds identified by the methods described herein.

[0036] In some other respects, this document provides a method for identifying a reservoir of β-catenin in cells, the method comprising: (a) contacting a composition containing cells with a first detectable reagent that specifically recognizes a β-catenin polypeptide; (b) contacting the composition containing cells with a second detectable reagent that specifically recognizes phosphorylated H2A.X (P-2A.X) or DNA damage checkpoint mediator 1 (MDC1); and (c) detecting the first and second detectable reagents; wherein the detection of the colocalization of the first and second detectable reagents within the cells identifies a reservoir of β-catenin in the cells.

[0037] Those skilled in the art will also understand that changes may be made to the form and details of the embodiments described herein without departing from the scope of this disclosure. Furthermore, although various advantages, aspects, and objectives have been described with reference to various embodiments, the scope of this disclosure should not be limited by such references to advantages, aspects, and objectives.

[0038] All references cited in this paper (including patent applications and publications) are incorporated herein by reference in their entirety.

[0039] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. For example, some aspects of this disclosure are presented in a modular manner, and this presentation should not be construed as limiting the possible combinations of methods taught herein.

[0040] I. Definition For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form and vice versa. If any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth therein shall prevail.

[0041] As used herein, “condensate” means a non-membrane-encapsulated compartment formed by the phase separation (including all stages of phase separation) of one or more proteins and / or other macromolecules (such as nucleic acids).

[0042] As used herein, the terms “peptide” and “protein” are used interchangeably to refer to polymers containing amino acid residues and are not limited to a minimum length. Such polymers may contain natural or non-natural amino acid residues or combinations thereof, and include, but are not limited to, peptides, polypeptides, oligopeptides, dimers, trimers, and polymers of amino acid residues. Full-length polypeptides or proteins and fragments thereof are covered in this definition. The term also includes the types of modifications, such as post-translational modifications of one or more residues, including but not limited to methylation, phosphorylation, glycosylation, sialylation, or acetylation.

[0043] The term "antibody" and its grammatical equivalents include full-length antibodies and their antigen-binding fragments. A full-length antibody comprises two heavy chains and two light chains. As used herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, biantibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized biantibodies (ds biantibodies), single-chain antibody molecules (scFv), scFv dimers (bivalent biantibodies), multispecific antibodies formed from portions of antibodies containing one or more CDRs, camelified single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, any other antibody fragment that binds to an antigen but does not contain the complete full-length antibody structure, or antibody mimics (e.g., designed ankylosing spicule repeats (DARPin), affinities, or monomers (ADNECTINS®)). The antigen-binding fragment can bind to the same antigen bound by the parent antibody or a fragment of the parent antibody (e.g., parental scFv).

[0044] As used herein, the terms “specific binding” or “specific to” or their grammatical equivalents refer to a measurable and reproducible interaction, such as the binding between a target and an antibody or antibody moiety, which determines the presence of the target in the presence of a heterogeneous group of molecules, including biomolecules. For example, an antibody or antibody moiety that specifically binds to a target (which may be an epitope) binds to that target with greater affinity, stronger affinity, easier binding, and / or longer duration than it binds to other targets. In some embodiments, an antibody or antibody moiety that specifically binds to an antigen reacts with one or more antigenic determinants of the antigen (e.g., β-catenin or a portion thereof, including β-catenin species containing post-translational modifications such as one or more phosphorylated β-catenins) with a binding affinity at least about 10 times its binding affinity to other targets.

[0045] As used herein, the terms “comprising,” “having,” “containing,” and “including,” and other similar forms and their grammatical equivalents, are intended to be synonymous and open-ended in meaning, because one or more items following any of these words do not imply an exhaustive list of those items or that the list is limited to only those items. For example, an article “comprising” components A, B, and C may consist of components A, B, and C (i.e., contain only), or may contain not only components A, B, and C, but also one or more other components. Therefore, it is intended and understood that “comprising” and its similar forms and grammatical equivalents include the disclosed content of embodiments “consisting substantially of” or “consisting of.”

[0046] When a range of values ​​is provided, it is understood that, unless the context clearly indicates otherwise, every intermediate value between the upper and lower limits of the range up to one-tenth of the lower limit unit, and any other specified value or intermediate value within the specified range, are included in this disclosure and are subject to any specific exclusions within the specified range. Where a specified range includes one or both of the included limits, the range excluding one or both of those included limits is also included in this disclosure.

[0047] References to “about” a value or parameter in this document include (and descriptions) variations of said value or parameter itself. For example, a description of “about X” includes a description of “X”.

[0048] As used herein (including in the appended claims), unless the context clearly specifies otherwise, the singular forms “a”, “or” and “the” include plural indicators.

[0049] II. Methods for determining β-catenin reservoirs and / or identifying β-catenin reservoir regulators In some respects, this document provides methods for determining a β-catenin reservoir of a composition comprising cells or a derivative thereof, and uses thereof, for example, for identifying β-catenin reservoir modulators (e.g., small molecule therapeutic compounds that modulate, for example, increasing the β-catenin reservoir in cells, compared to a reference).

[0050] In some aspects, this document provides a method for identifying (e.g., visualizing and / or measuring) a β-catenin reservoir in a composition containing cells, the method comprising contacting the composition containing cells or derivatives thereof with a detectable reagent (e.g., an antibody or an antigen-binding fragment thereof) that specifically binds to macromolecules in the β-catenin reservoir (e.g., allowing sufficient signal against baseline noise values ​​to image the β-catenin reservoir). As described herein, a detectable reagent that specifically binds to macromolecules in a β-catenin reservoir is a detectable reagent that identifies the presence of a target in the presence of a heterogeneous molecular population. In some embodiments, the detectable reagent may also bind other molecules, including macromolecules (e.g., biomolecules), to achieve a degree of affinity and / or affinity. In some embodiments, the macromolecules are also located external to the β-catenin reservoir. In some embodiments, the macromolecules are peptides or similar types, such as peptides characterized by one or more specific post-translational modifications (e.g., as defined by peptide attachment sites and / or composition). In some embodiments, the peptide contains one or more phosphorylations. In some embodiments, the macromolecule is a β-catenin polypeptide, for example, containing one or more phosphorylated inactive β-catenin polypeptides at one or more of S33, S37, T41, or S45, and wherein the amino acid positions are relative to the amino acid sequence of SEQ ID NO:1. In some embodiments, a β-catenin reservoir is identified, for example, by detecting inactive β-catenin polypeptides (e.g., containing one or more phosphorylations at one or more of S33, S37, T41, or S45) above a threshold level using a detectable reagent. In some embodiments, the threshold level is background noise (e.g., fluorescence noise signal from immunofluorescence staining or imaging). In some embodiments, the threshold level is the average or median fluorescence signal in non-aggregates (e.g., around aggregates) or in the cytoplasm. As described herein, general techniques for condensate identification are known in the art (e.g., as described in 2020 / 0150107, WO 2022 / 035989, and WO 2022 / 187225, which are incorporated herein by reference) and can be combined with the teachings herein to identify β-catenin reservoirs. In some embodiments, the inactive β-catenin polypeptide contains phosphorylation at T41 and / or S45 (including T41 and S45), wherein the amino acid positions are relative to the amino acid sequence of SEQ ID NO:1. In some embodiments, the detectable reagent comprises a 23H16L13 antibody clone or a fragment thereof having substantially the same binding specificity as the 23H16L13 antibody clone. In some embodiments, the detectable reagent comprises an antibody or an antigen-binding fragment thereof comprising all six CDRs of 23H16L13, such as a humanized form of the 23H16L13 antibody clone.In some embodiments, the macromolecule is phosphorylated H2A.X (P-2A.X). In some embodiments, the macromolecule is DNA damage checkpoint protein 1 (MDC1). In some embodiments, the method includes detecting more than one macromolecule, such as one or more inactive β-catenin peptides, P-2A.X, or MDC1. In some embodiments, the method includes detecting the co-localization of more than one macromolecule, such as two or more of any inactive β-catenin peptides, P-2A.X, or MDC1.

[0051] In some aspects, this document provides a method for identifying β-catenin reservoirs in cells, the method comprising: (a) contacting a cell-containing composition with a detectable reagent (e.g., an antibody or an antigen-binding fragment thereof), the detectable reagent specifically recognizing an inactive form of β-catenin polypeptide containing phosphorylation at T41 and / or S45; and (b) detecting the detectable reagent, wherein detecting the detectable reagent intracellularly (e.g., in aggregates) to identify β-catenin reservoirs in cells. In some embodiments, the detectable reagent is an antibody that specifically recognizes an inactive form of β-catenin polypeptide containing phosphorylation at T41 and / or S45, including phosphorylation at both T41 and S45. In some embodiments, the detectable reagent comprises a 23H16L13 antibody clone or a fragment thereof having substantially the same binding specificity as the 23H16L13 antibody clone. In some implementations, the detectable reagent includes an antibody or an antigen-binding fragment thereof containing all six CDRs of 23H16L13, such as a humanized form of a 23H16L13 antibody clone.

[0052] In some respects, this document provides a method for identifying β-catenin reservoirs in cells, the method comprising: (a) contacting a cell-containing composition with a detectable reagent (e.g., an antibody or an antigen-binding fragment thereof) that specifically recognizes an inactive form of a β-catenin polypeptide (e.g., containing phosphorylation at one or more of S33, S37, T41, or S45); (b) detecting aggregates or components thereof (e.g., by contacting the cell-containing composition with a second detectable reagent (e.g., an antibody or an antigen-binding fragment thereof) that specifically recognizes aggregate components, or using fluorescently labeled aggregate components); and (c) detecting the detectable reagent and aggregates or components thereof; wherein the detection of colocalization of the detectable reagent and aggregates or components thereof within cells identifies β-catenin reservoirs in cells.

[0053] In some aspects, this document provides a method for identifying β-catenin reservoirs in cells, the method comprising: (a) contacting a cell-containing composition with a detectable reagent (e.g., an antibody or an antigen-binding fragment thereof), said detectable reagent specifically recognizing an inactive form of a β-catenin polypeptide (e.g., containing phosphorylation at one or more of S33, S37, T41, or S45); (b) detecting an agglomerate or a component thereof (e.g., by contacting the cell-containing composition with a second detectable reagent (e.g., an antibody or an antigen-binding fragment thereof) that specifically recognizes an agglomerate component, or using a fluorescently labeled agglomerate component); and (c) detecting the detectable reagent and the agglomerate or a component thereof; wherein the detection of co-localization of the detectable reagent and the agglomerate or a component thereof within the cell identifies β-catenin reservoirs in cells. In some embodiments, the agglomerates are associated with long-term DSB DNA damage. In some embodiments, the agglomerates associated with long-term DSB DNA damage comprise P-2A.X and / or MDC1. In some embodiments, the agglomerate component to be detected is P-2A.X and / or MDC1. In some embodiments, the condensates associated with long-term DSB DNA damage also contain one or more of 53BP1, RIF1, p-TEFb, DCP2, or FOXO3a. Therefore, in some embodiments, the condensate component to be detected may be one or more of 53BP1, RIF1, p-TEFb, DCP2, or FOXO3a.

[0054] In some respects, this document provides a method for identifying β-catenin reservoirs in cells, the method comprising: (a) contacting a cell-containing composition with a detectable reagent (e.g., an antibody or an antigen-binding fragment thereof), the detectable reagent specifically recognizing P-2A.X; and (b) detecting the detectable reagent, wherein detecting the detectable reagent in cells (e.g., within aggregates) to identify β-catenin reservoirs in cells.

[0055] In some respects, this document provides a method for identifying β-catenin reservoirs in cells, the method comprising: (a) contacting a cell-containing composition with a detectable reagent (e.g., an antibody or an antigen-binding fragment thereof), the detectable reagent specifically recognizing MDC1; and (b) detecting the detectable reagent, wherein detecting the detectable reagent intracellularly (e.g., within an aggregate) to identify β-catenin reservoirs in cells.

[0056] In some aspects, this document provides a method for identifying β-catenin reservoirs in cells, the method comprising: (a) contacting a cell-containing composition with a first detectable reagent (e.g., an antibody or an antigen-binding fragment thereof) that specifically recognizes a β-catenin polypeptide; (b) contacting the cell-containing composition with a second detectable reagent (e.g., an antibody or an antigen-binding fragment thereof) that specifically recognizes P-2A.X or MDC1; and (c) detecting the first and second detectable reagents; wherein the co-localization of the first and second detectable reagents within the cells identifies the β-catenin reservoir in the cells. As described herein, the identification of β-catenin reservoirs is assessed by observing regions of higher intensity signals compared to background and / or threshold. The first detectable reagent is capable of specifically recognizing any form of β-catenin polypeptide, such as the active form, inactive form, or pan-β-catenin. In some embodiments, the first detectable reagent is an antibody that specifically recognizes the inactive form of the β-catenin polypeptide, comprising phosphorylation at T41 and / or S45, including phosphorylation at T41 and S45. In some embodiments, the first detectable reagent comprises a 23H16L13 antibody clone or a fragment thereof, the fragment having substantially the same binding specificity as the 23H16L13 antibody clone. In some embodiments, the first detectable reagent comprises an antibody or an antigen-binding fragment thereof containing all six CDRs of 23H16L13, such as a humanized form of the 23H16L13 antibody clone. In some embodiments, the first detectable reagent comprises an antibody or an antigen-binding fragment thereof that recognizes a β-catenin polypeptide, regardless of its phosphorylation state.

[0057] In some aspects, this document provides a method for identifying compounds that regulate cellular β-catenin reservoirs (e.g., any β-catenin reservoir identified herein, or any β-catenin reservoir identified by any method described herein), the method comprising: (a) contacting a cell-containing composition with the compound; and (b) identifying the compound regulating the cellular β-catenin reservoir by comparing the cellular β-catenin reservoir with a reference. In some embodiments, the method further includes the determination of the cellular β-catenin reservoir, for example using any β-catenin reservoir identification method described herein. Thus, in some aspects, this document provides a method for identifying compounds capable of regulating intracellular β-catenin reservoirs, the method comprising: (a) contacting a cell-containing composition with the compound; (b) determining the cellular β-catenin reservoir of the cell-containing composition or a derivative thereof; and (c) identifying the compound regulating the cellular β-catenin reservoir by comparing the cellular β-catenin reservoir determined to be a reference. In some embodiments, the assay of a β-catenin reservoir includes detecting an inactive form of the β-catenin peptide using a detectable reagent. In some embodiments, the detectable reagent is an antibody that specifically recognizes an inactive form of the β-catenin peptide, comprising phosphorylation at T41 and / or S45. In some embodiments, the detectable reagent comprises a 23H16L13 antibody clone or a fragment thereof, having substantially the same binding specificity as the 23H16L13 antibody clone. In some embodiments, the detectable reagent comprises an antibody or an antigen-binding fragment thereof comprising all six CDRs of 23H16L13, such as a humanized form of the 23H16L13 antibody clone. In some embodiments, the assay of a β-catenin reservoir includes detecting the co-localization of the β-catenin peptide (e.g., any form of β-catenin peptide) and P-2A.X and / or MDC1. In some embodiments, comparing a β-catenin reservoir measured in cells with a reference is based on one or more of the following properties of the β-catenin reservoir: (i) the presence of the β-catenin reservoir; (ii) the number of β-catenin reservoirs; (iii) the size of the β-catenin reservoir; (iv) the surface area of ​​the β-catenin reservoir; (v) the cellular localization of the β-catenin reservoir; (vi) the distribution of the β-catenin reservoir; (vii) the ratio of the amount of the β-catenin reservoir to the amount of the reference aggregate; or (viii) the composition of the β-catenin reservoir. In some embodiments, regulation of the β-catenin reservoir is based on an increase in the number of β-catenin reservoirs in cells compared to a reference. In some embodiments, regulation of the β-catenin reservoir is based on an increase in the number of β-catenin reservoirs in cells compared to a reference.In some embodiments, the regulation of cellular β-catenin reservoirs is based on an increase in the number of β-catenin reservoirs in the cell nucleus compared to a reference. In some embodiments, the regulation of cellular β-catenin reservoirs is based on an increase in the presence of β-catenin reservoirs in the cell compared to a reference. In some embodiments, the regulation of cellular β-catenin reservoirs is based on an increase in the presence of β-catenin reservoirs in the cell nucleus compared to a reference.

[0058] In some aspects described herein, “β-catenin reservoir” or its grammatical equivalents refer to a nuclear condensate characterized by the presence of one or more inactive forms of β-catenin. In some embodiments, one or more inactive forms of β-catenin are associated with reduced or absent transcriptional activity compared to the active form of β-catenin (e.g., β-catenin phosphorylated at S552 and / or S675). In some embodiments, the inactive form of β-catenin comprises phosphorylation at one or more of S33, S37, T41, or S45. In some embodiments, the inactive form of β-catenin comprises phosphorylation at both T41 and S45. In some embodiments, the inactive form of β-catenin is not phosphorylated at S552 and / or S675. See, for example, Verheyen & Gottardi, Dev Dyn, 239, 2010. In some embodiments, the β-catenin reservoir further comprises one or more active forms of β-catenin. In some embodiments, the β-catenin reservoir further comprises another macromolecule, such as a polypeptide or a specific type thereof, such as a type of post-translational modification. In some embodiments, the β-catenin reservoir selectively comprises another macromolecule, for example, that is not present in another condensate, or that is not present in another condensate containing β-catenin (e.g., the active form of β-catenin). In some embodiments, the β-catenin reservoir is not a transcriptional condensate. In some embodiments, the β-catenin reservoir does not contain one or more mediator components. In some embodiments, the β-catenin reservoir does not contain DNA and / or RNA. In some embodiments, the β-catenin reservoir is not present at the transcription start site of a gene. In some embodiments, the presence of the β-catenin reservoir indicates a specific biological function desired to be provided by a small molecule therapeutic compound, such as reduced activity of β-catenin-related transcription compared to the active form of β-catenin. In some embodiments, the formation (including increase) of the β-catenin reservoir is not associated with increased cell death. In some embodiments, transcription factors LEF1 and / or TCF4 fail to rescue the β-catenin reservoir (releasing inactive β-catenin from the reservoir to convert it into the active form). In some embodiments, the β-catenin reservoir is not associated with closed chromatin. In some embodiments, the β-catenin reservoir does not contain PRMT5.

[0059] This application (including the following sections) describes in more detail the methods, elements, and uses for determining the β-catenin reservoir. The modular discussion of these methods and elements is not intended to limit the scope of the teachings provided herein, and those skilled in the art will readily understand their combination based on the descriptions provided herein.

[0060] A. Determination and detection of β-catenin reservoir In some aspects of the methods described herein, the methods include determining a composition comprising cells or a derivative thereof to detect a β-catenin reservoir (or its absence, such as a submeasurable amount). As described herein, the determination of a β-catenin reservoir includes detecting components of the β-catenin reservoir (or its absence), such as macromolecules, such as peptides, or nucleic acids, such as DNA or RNA. It will be readily understood by those skilled in the art that many techniques can be used to perform such determinations, and the following description of exemplary techniques is not intended to limit the scope of the teachings provided herein.

[0061] In some embodiments, determining a β-catenin reservoir includes detecting an inactive form of the β-catenin peptide. In some embodiments, detecting an inactive form of the β-catenin peptide includes contacting a cell-containing composition or a derivative of that composition with a detectable reagent that specifically recognizes the inactive form of the β-catenin peptide. In some embodiments, the detectable reagent is an antibody that specifically recognizes an inactive form of the β-catenin peptide. In some embodiments, the determination includes detecting the detectable reagent that specifically recognizes the inactive form of the β-catenin peptide. In some embodiments, the detectable reagent that specifically recognizes the inactive form of the β-catenin peptide is a labeled (e.g., fluorescent or colorimetric, e.g., enzyme-based) antibody (e.g., a labeled primary antibody) that specifically recognizes the inactive form of the β-catenin peptide. In some embodiments, a labeled (e.g., fluorescent or enzyme-catalyzed) secondary antibody that specifically recognizes the detectable reagent is used to detect the detectable reagent that specifically recognizes the inactive form of the β-catenin peptide. In some embodiments, the detectable reagent that specifically recognizes the inactive form of the β-catenin polypeptide specifically recognizes phosphorylation at one or more of S33, S37, T41, or S45 of the inactive form of the β-catenin polypeptide, wherein the amino acid positions are relative to the amino acid sequence of SEQ ID NO:1 (Table 1). In some embodiments, the detectable reagent that specifically recognizes the inactive form of the β-catenin polypeptide specifically recognizes phosphorylation at T41 and / or S45. In some embodiments, the detectable reagent that specifically recognizes the inactive form of the β-catenin polypeptide specifically recognizes phosphorylation at both T41 and S45. In some embodiments, the detectable reagent that specifically recognizes the inactive form of the β-catenin polypeptide is a 23H16L13 antibody clone or a humanized form thereof.

[0062] In some embodiments, determining the β-catenin reservoir includes (including further including) detecting an active form of the β-catenin polypeptide (or its absence), such as a β-catenin polypeptide containing one or more C-terminal phosphorylated β-catenin polypeptides. In some embodiments, determining the β-catenin reservoir includes (including further including) detecting an active form of the β-catenin polypeptide (or its absence), such as a β-catenin polypeptide phosphorylated at sites S552 and / or S675, wherein the amino acid positions are relative to the amino acid sequence of SEQ ID NO:1. In some embodiments, the detectable reagent that specifically recognizes the active form of the β-catenin polypeptide is a labeled (e.g., fluorescently labeled or enzyme-labeled) antibody (e.g., a labeled primary antibody) that specifically recognizes the active form of the β-catenin polypeptide. In some embodiments, the detectable reagent that specifically recognizes the active form of the β-catenin polypeptide is detected using a fluorescently labeled secondary antibody that specifically recognizes said detectable reagent.

[0063] In some embodiments, determining the β-catenin reservoir includes (including further including) detecting phosphorylated H2A.X (P-2A.X) using a detectable reagent. In some embodiments, the detectable reagent that specifically recognizes P-2A.X is a labeled (e.g., fluorescently labeled or enzyme-labeled) antibody that specifically recognizes P-2A.X (e.g., a labeled primary antibody). In some embodiments, the detectable reagent that specifically recognizes P-2A.X is detected using a labeled (e.g., fluorescently labeled or enzyme-labeled) secondary antibody that specifically recognizes the detectable reagent. Any anti-P-2A.X antibody or its antigen-binding fragment, such as a 20E3 antibody clone, may be used herein.

[0064] In some embodiments, determining the β-catenin reservoir includes (including further including) detecting DNA damage checkpoint mediator 1 (MDC1) using a detectable reagent. In some embodiments, the detectable reagent that specifically recognizes MDC1 is a labeled (e.g., fluorescent or enzyme-catalyzed) antibody (e.g., a labeled primary antibody) that specifically recognizes MDC1. In some embodiments, a labeled (e.g., fluorescent or enzyme-catalyzed) secondary antibody that specifically recognizes the detectable reagent is used to detect the detectable reagent that specifically recognizes MDC1. Any anti-MDC1 antibody or its antigen-binding fragment, such as P2B11 or MDC1-50 antibody clones, may be used herein.

[0065] In some embodiments, determining a β-catenin reservoir includes (including further including) detecting one or more macromolecules using a detectable reagent: p53-binding protein 1 (53BP1), RIF1, positive transcription elongation factor (p-TEFb), mRNA decapping enzyme 2 (DCP2), and FOXO3a. In some embodiments, the detectable reagent that specifically recognizes such a macromolecule is a labeled (e.g., fluorescent or enzymatic) antibody (e.g., a labeled primary antibody) that specifically recognizes such a macromolecule. In some embodiments, a labeled (e.g., fluorescent or enzymatic) secondary antibody that specifically recognizes the detectable reagent is used to detect the detectable reagent that specifically recognizes such a macromolecule.

[0066] In some embodiments, determining a β-catenin reservoir includes (including further including) detecting a β-catenin polypeptide (or its absence), such as any form of β-catenin polypeptide, such as non-phosphorylated β-catenin, N-terminal phosphorylated β-catenin (e.g., phosphorylated at one or more of S33, S37, T41, or S45), C-terminal phosphorylated β-catenin (e.g., phosphorylated at S552 and / or S675), or any combination thereof. In some embodiments, determining a β-catenin reservoir includes detecting the co-localization (or absence) of any form of β-catenin polypeptide with a macromolecule (e.g., MDC1 and / or P-2A.X). In some embodiments, determining a β-catenin reservoir further includes detecting the co-localization (or absence) of any of 53BP1, RIF1, p-TEFb, DCP2, or FOXO3a with the co-localization signal of the β-catenin polypeptide and MDC1 and / or P-2A.X. Antibodies that can specifically bind to various forms of β-catenin or their antigen-binding fragments are well known in the art, including but not limited to the 12F7 antibody clone.

[0067] In some embodiments, the detectable reagent includes a label such as a fluorescent label, for example, GFP or mCherry. In some embodiments, the detectable reagent includes a detectable feature inherent to the detectable reagent, such as a fluorophore that is an inherent feature of the detectable reagent. In some embodiments, the detectable reagent comprises an antibody, aptamer, or nucleic acid for the indirect detection and / or direct measurement of macromolecules in a β-catenin reservoir. In some embodiments, the detectable reagent includes a radiolabel, a colorimetric label, a luminescent label, a chemically reactive label (e.g., a component portion used in click chemistry), or a fluorescent label.

[0068] In some embodiments, the macromolecule (e.g., a polypeptide or nucleic acid, such as DNA or RNA) of the β-catenin reservoir used for detection is a fusion molecule containing a label (e.g., a fluorescent label). In some embodiments, the fusion molecule includes Halo, Dendra2, GFP, RFP, or mCherry moieties. In some embodiments, the macromolecule of the β-catenin reservoir used for detection contains detectable features inherent to the macromolecule.

[0069] In some embodiments, the detection reagent is not a fusion protein, for example, not a fusion macromolecule associated with a β-catenin reservoir. In some embodiments, the detection reagent directly binds to a β-catenin peptide, such as an inactive form of the β-catenin peptide.

[0070] Those skilled in the art will readily understand the assay and detection methods described herein, for example, using immunofluorescence (IF) staining techniques. In some embodiments, the method includes detecting more than one macromolecule, wherein the method includes using more than one detectable reagent (e.g., one detectable reagent for each macromolecule). In some embodiments, the detectable reagents can be detected by imaging techniques (e.g., microscopy, such as fluorescence microscopy). In some embodiments, more than one detectable reagent is configured for simultaneous use (e.g., a cell-containing composition or a derivative of the composition is contacted with a first detectable reagent and a second detectable reagent, wherein the first and second detectable reagents specifically bind to different biomolecule species). In some embodiments, more than one detectable reagent is configured for serial use (e.g., a cell-containing composition or a derivative of the composition is contacted with a first detectable reagent, which is removed before the composition or derivative of the composition is contacted with a second detectable reagent, wherein the first and second detectable reagents specifically bind to different biomolecule species). In some embodiments, when more than one detectable reagent is configured for serial use, the cell-containing composition or a derivative thereof is contacted with a first detectable reagent, and then with a second detectable reagent, without removing the first detectable reagent. In some embodiments, when evaluating more than one macromolecule in a β-catenin reservoir, it is helpful to utilize antibodies from different species, such as mouse antibodies specific to the first macromolecule and rabbit antibodies specific to the second macromolecule. Such methods achieve co-staining. In some embodiments, different labeling techniques or co-staining are used to achieve co-localization measurements. In some embodiments, co-localization measurements are performed by overlaying images, where each image includes staining of one or more aspects. In some embodiments, the detection method (e.g., IF staining technique) includes washing away unbound detectable reagents prior to imaging.

[0071] In some embodiments, detection includes imaging at least a portion of a cell, such as imaging a specific cell localization (e.g., the nucleus or a portion thereof) or imaging a field of view containing at least a portion of a cell. In some embodiments, imaging includes fluorescence imaging techniques. In some embodiments, imaging techniques include any one or more of the following: immunofluorescence (IF), in situ hybridization (ISH, such as FISH), gene fusion (e.g., GFP labeling), or dyes specific to macromolecules of β-catenin reservoirs. In some embodiments, imaging techniques include using reagents to visualize cellular features such as the cell membrane or organelles such as the nucleus (e.g., by DAPI staining). In some embodiments, the methods provided herein also include measuring (e.g., quantifying) the signal of a detectable reagent.

[0072] In some embodiments, the imaging techniques include the use of microscopy techniques (and associated microscopic instruments). In some embodiments, the microscopy techniques include confocal microscopy. In some embodiments, the microscopy techniques include fluorescence microscopy. In some embodiments, the microscopy techniques include high-resolution microscopy. In some embodiments, the microscopy techniques include stimulated emission depletion (STED) microscopy. In some embodiments, the microscopy techniques include SoRa super-resolution rotating disk microscopy. In some embodiments, the microscopy techniques include electron microscopy techniques (such as cryo-EM or cryo-ET). In some embodiments, the microscopy techniques include total internal reflection fluorescence (TIRF) microscopy.

[0073] In some embodiments, the method includes assessing the macromolecular distribution of β-catenin reservoirs. In some embodiments, the macromolecular distribution is based on pixel analysis. In some embodiments, pixel analysis is performed on regions of the cell, such as the nucleus and / or cytoplasm or regions thereof. In some embodiments, pixel analysis is performed without identifying individual aggregates or their characteristics, such as aggregate boundaries. For example, pixels with signals above a threshold (e.g., above a background level) indicate the presence of aggregates, such as β-catenin reservoirs. In some embodiments, aggregates are identified using a different reagent (e.g., a dye) specifically dispensed into the aggregates. Pixels used for the pixel analysis described herein can be of any shape and size relative to constraints imposed on the analyzed region. For example, in some embodiments, the analyzed region contains at least two pixels, such as any of at least 10, 100, 1,000, 5,000, 10,000, 20,000, 30,000, 40,000, or 50,000 pixels, such as within the boundaries of the cell nucleus or cytoplasm in a cell image. In some embodiments, the pixel size is uniform. In some implementations, the pixel shape is uniform.

[0074] In some embodiments, the methods provided herein further include contacting the cell-containing composition or a derivative thereof with a fixative (e.g., formaldehyde) prior to performing an assay or detection step. In some embodiments, the methods provided herein further include determining one or more cellular characteristics of the cells, for example by staining the cells with a dye.

[0075] In some embodiments, the methods provided herein further include subjecting the cell-containing composition to conditions that promote β-catenin reservoir formation, such as in compound screening assays, prior to contacting the cell-containing composition with the compound. In some embodiments, β-catenin reservoir formation includes expressing a β-catenin polypeptide in the cells (e.g., at a level greater than that observed endogenously in cells or populations thereof). In some embodiments, conditions that promote β-catenin reservoir formation include activating or upregulating Wnt signaling in the cells, such as inducing nuclear translocation of β-catenin.

[0076] Those skilled in the art will readily understand the scope of the detectable reagents covered herein. For example, techniques for evaluating whether a reagent is a detectable reagent suitable for the methods described herein are well known in the art, such as detectable reagents that specifically recognize inactive forms of β-catenin peptides. In some embodiments, ELISA or cell models with well-characterized β-catenin reservoir phenotypes may be used to confirm that a reagent is a detectable reagent suitable for the methods described herein.

[0077] B. Comparison and identification of β-catenin reservoir regulators In some embodiments, the methods provided herein include comparing a β-catenin reservoir between a cell-containing assay composition or a derivative thereof and a reference. For example, in some aspects, a method is provided herein for identifying compounds that regulate β-catenin reservoirs in cells, the method comprising: identifying compounds that regulate β-catenin reservoirs in cells by comparing β-catenin reservoirs measured in cells with a reference. In some embodiments, detecting β-catenin reservoirs in cells treated with a compound (e.g., their presence) identifies the compound as a β-catenin reservoir regulating cells.

[0078] In some embodiments, comparing a β-catenin reservoir measured in a cell with a reference is based on one or more of the following properties of the β-catenin reservoir: (i) the presence (including its absence); (ii) the number of β-catenin reservoirs (including cell-localized counts, such as β-catenin reservoirs in the nucleus); (iii) the size of the β-catenin reservoir; (iv) the surface area of ​​the β-catenin reservoir; (v) the cellular localization of the β-catenin reservoir; (vi) the distribution of the β-catenin reservoir; (vii) the ratio of the amount of the β-catenin reservoir to the amount of the reference aggregate; or (viii) the composition of the β-catenin reservoir. In some embodiments, the size of the β-catenin reservoir is assessed by one or more of the following: maximum span dimension (e.g., diameter), perimeter (e.g., circumference), cross-sectional area (e.g., maximum cross-sectional area), or volume.

[0079] In some embodiments, comparing a β-catenin reservoir measured in cells with a reference is based on (including further based on) one or more of the following properties: (viii) functional activity associated with one or more β-catenin reservoirs; (ix) composition of one or more β-catenin reservoirs; (x) colocalization of one or more β-catenin reservoirs with macromolecules; (xi) diffusion coefficient of the components of one or more β-catenin reservoirs; (xii) stability of one or more β-catenin reservoirs; (xiii) sphericity of one or more β-catenin reservoirs; (xiv) fluidity of one or more β-catenin reservoirs; or (xv) solidification of one or more β-catenin reservoirs.

[0080] In summary, the methods described herein are capable of identifying one or more compounds that regulate β-catenin reservoirs in a desired manner. In some embodiments, the regulation of β-catenin reservoirs is based on an increase in the number of β-catenin reservoirs in cells compared to a reference. In some embodiments, the regulation of β-catenin reservoirs in cells is based on an increase in the number of β-catenin reservoirs in the nucleus compared to a reference. In some embodiments, the regulation of β-catenin reservoirs in cells is based on an increase in the presence of β-catenin reservoirs in cells compared to a reference. In some embodiments, the regulation of β-catenin reservoirs in cells is based on an increase in the presence of β-catenin reservoirs in the nucleus compared to a reference.

[0081] In some aspects of the methods presented herein, the characterization (including further evaluation) of cellular β-catenin reservoirs can be assessed using macromolecules, such as those associated with β-catenin reservoirs. The assays and detection methods described above can also be used to assess such macromolecules. Furthermore, other techniques can be used to assess such macromolecules, including protein measurement techniques such as Western blotting, ELISA, and mass spectrometry. In some embodiments, comparing a β-catenin reservoir measured in cells with a reference is based on (including further based on) one or more of the following properties of the inactive form of the β-catenin polypeptide associated with the β-catenin reservoir: (i) the localization of the inactive form of the β-catenin polypeptide; (ii) the amount of the inactive form of the β-catenin polypeptide; (iii) the distribution of the inactive form of the β-catenin polypeptide; (iv) the aggregate partitioning of the inactive form of the β-catenin polypeptide in the β-catenin reservoir and / or one or more other aggregates; (v) the functional activity associated with the inactive form of the β-catenin polypeptide; (vi) the aggregation of the inactive form of the β-catenin polypeptide; (vii) the post-translational modification state of the inactive form of the β-catenin polypeptide; or (viii) the amount of degradation products of the inactive form of the β-catenin polypeptide.In some embodiments, the regulation of β-catenin reservoirs in cells, compared to reference cells, is based on one or more of the following: (i) an increase in the amount of phosphorylated β-catenin peptides at one or more of S33, S37, T41, and S45 in the cells; (ii) the presence of phosphorylated β-catenin peptides at one or more of S33, S37, T41, and S45 in the cells; (iii) an increase in the amount of phosphorylated β-catenin peptides at one or more of S33, S37, T41, and S45 in one or more aggregates (e.g., β-catenin reservoirs, or aggregates associated with persistent double-strand breaks (DSB) DNA damage); (iv) the presence of S33, S37, T41, and S45 in one or more aggregates (e.g., β-catenin reservoirs or aggregates associated with persistent double-strand breaks (DSB) DNA damage). (v) A decrease in the amount of β-catenin peptide phosphorylated at S552 and / or S675 sites in a cell (e.g., a cell nucleus); (vi) Absence of β-catenin peptide phosphorylated at S552 and / or S675 sites in a cell (e.g., a cell nucleus); (vii) A decrease in the amount of β-catenin peptide phosphorylated at S552 and / or S675 sites in one or more condensates; (viii) Absence of β-catenin peptide phosphorylated at S552 and / or S675 sites in one or more condensates; (ix) An increase in the amount of β-catenin peptide phosphorylated at S552 and / or S675 sites in one or more β-catenin reservoirs; and (x) The presence of β-catenin peptide phosphorylated at S552 and / or S675 sites in one or more β-catenin reservoirs. In some embodiments, the regulation of the cellular β-catenin reservoir, compared to a reference, is based on an increase in the amount of inactive β-catenin polypeptide in the reservoir compared to a reference. In some embodiments, the inactive β-catenin polypeptide is a wild-type β-catenin polypeptide. In some embodiments, the inactive β-catenin polypeptide is a mutant β-catenin polypeptide.

[0082] In some embodiments, comparing the β-catenin reservoir measured in cells with a reference is based on (including further based on): i) one or more properties of β-catenin polypeptides (e.g., any form of β-catenin polypeptide, such as β-catenin polypeptides associated with β-catenin reservoirs) and ii) one or more properties of macromolecules (e.g., MDC1 and / or P-2A.X). Such one or more properties include: (i) the localization of β-catenin peptides and / or macromolecules; (ii) the amount of β-catenin peptides and / or macromolecules; (iii) the distribution of β-catenin peptides and / or macromolecules; (iv) the aggregate distribution of β-catenin peptides and / or macromolecules in β-catenin reservoirs and / or one or more other aggregates; (v) the functional activity associated with β-catenin peptides and / or macromolecules; (vi) the aggregation of β-catenin peptides and / or macromolecules; (vii) the post-translational modification state of β-catenin peptides and / or macromolecules; or (viii) the amount of degradation products of β-catenin peptides and / or macromolecules.

[0083] In some embodiments, the regulation of cellular β-catenin reservoirs compared to a reference is based on an increase in active β-catenin isolated into the reservoirs. In some embodiments, the regulation of cellular β-catenin reservoirs compared to a reference is based on a decrease in condensates of active β-catenin, such as those containing one or more mediators or active transcription factors (e.g., active Pol II), within transcriptional active condensates. In some embodiments, the active form of the β-catenin polypeptide is a wild-type β-catenin polypeptide. In some embodiments, the active form of the β-catenin polypeptide is a mutant β-catenin polypeptide.

[0084] In some embodiments, the regulation of β-catenin reservoirs in cells, compared to a reference, is based on: (i) an increase in the amount of one or more phosphorylated β-catenin polypeptides at S33, S37, T41, and S45 in one or more aggregates; and (ii) the presence of phosphorylated β-catenin polypeptides at one or more of S33, S37, T41, and S45 in one or more aggregates. In some embodiments, one or more aggregates are β-catenin reservoirs, such as those identified using any of the methods described herein. In some embodiments, one or more aggregates are aggregates that are not bound to any β-catenin prior to contact of the cell-containing composition with the compound. In some embodiments, one or more aggregates are aggregates that are not associated with inactive forms of β-catenin prior to contact of the cell-containing composition with the compound (e.g., associated with phosphorylation at one or more of S33, S37, T41, and S45). In some embodiments, prior to contact of the cell-containing composition with the compound, one or more agglomerates are agglomerates associated with an active form of β-catenin (e.g., β-catenin polypeptides phosphorylated at S552 and / or S675) or a non-phosphorylated form of β-catenin. In some embodiments, one or more agglomerates are associated with long-term DSB DNA damage. In some embodiments, one or more agglomerates associated with long-term DSB DNA damage comprise P-2A.X and / or MDC1. In some embodiments, one or more agglomerates associated with long-term DSB DNA damage also comprise one or more of 53BP1, RIF1, p-TEFb, DCP2, or FOXO3a. Therefore, in some embodiments, any of the methods described herein, such as determining a β-catenin reservoir, determining the colocalization of β-catenin and macromolecules, determining the colocalization of β-catenin and agglomerates, and / or identifying a β-catenin reservoir, may include (including further including) detection with a detectable reagent that specifically recognizes one or more condensates described herein (e.g., those associated with long-term DSB DNA damage) or components thereof. For example, methods for detecting condensates and their components are described in U.S. Patents US 2020 / 0150107, WO 2022 / 035989, and WO 2022 / 187225, which are incorporated herein by reference.

[0085] In some embodiments, the reference is based on: (i) a composition comprising cells not in contact with the compound; (ii) a composition comprising cells mixed with a control medium and not in contact with the compound; (iii) a desired β-catenin profile (including theoretical profiles, e.g., no nuclear β-catenin reservoir, or a nuclear β-catenin reservoir profile induced by the desired control compound); (iv) a reference condensate or reference polypeptide within the cell (e.g., condensates not containing β-catenin and / or not regulated by the compound); and / or (v) a reference composition comprising cells (e.g., drug-resistant cell lines, such as cancer cell lines, or healthy cells without disease such as cancer). In some embodiments, the compound identified using any of the methods described herein can regulate the β-catenin reservoir in diseased cells (e.g., cells with aberrant Wnt / β-catenin signaling, such as cancer cells) but not in healthy cells.

[0086] In some embodiments, the comparisons described herein include comparing the signals of one or more detectable reagents used to measure β-catenin reservoirs in compound-treated cells with a reference. In some embodiments, characteristics of each cell are calculated, and an average is taken for the number of cells detected, for example, the average number of nuclear β-catenin reservoirs per cell. In some embodiments, the method includes determining a Z factor (Z') to score the regulation of β-catenin reservoirs in a composition containing cells. The use of the Z factor (Z') in high-throughput screening is well known in the art, for example, Zhang & Oldenburg, Z-Factor; Encyclopedia of Cancer , pp 3227-3228, 2008.

[0087] C. Cell-containing compositions and assay methods In some respects, the methods provided herein involve one or more compositions comprising cells (also referred to herein as cell compositions), assay forms, and their elements.

[0088] 1. Cell composition In some aspects, the methods described herein relate to one or more cell compositions. The cell composition may comprise any cell type or a mixture of any cell types. In some embodiments, the composition comprises cells. In some embodiments, the cells are a model of abnormal Wnt / β-catenin signaling. In some embodiments, the cells are a model of proliferative pathology. In some embodiments, the cells are a model of cancer. In some embodiments, the cells are cancer cells. In some embodiments, the cancer cells are human osteosarcoma U2OS cells or human colon adenocarcinoma DLD-1 cells. In some embodiments, the cells include APC truncation. In some embodiments, the cells are a model of autosomal dominant polycystic kidney disease (ADPKD), hereditary familial adenomatous polyposis (FAP), Alzheimer's disease, or metabolic diseases such as obesity and / or insulin resistance.

[0089] In some embodiments, the composition comprises at least one cell (e.g., the method described herein is a single-cell assay). In some embodiments, the composition comprises at least about 100 cells of a cell type, such as at least about 1,000 cells, 5,000 cells, or 1 x 10⁻⁶ cells. 4 1 cell, 5x10 4 1 cell, 1x10 5 1 cell, 5x10 5 One cell or 1x10 6 Any of the following in a cell.

[0090] In some embodiments, the composition comprises a single cell type. In some embodiments, the composition comprises at least about 1,000 cells of a single cell type. In some embodiments, the composition comprises multiple cell types, such as any one of two, three, four, or five cell types. In some embodiments, the composition is aliquoted for use in the method described herein, such as aliquots containing at least about 100 cells, such as any one of at least about 200 cells, 300 cells, 400 cells, 500 cells, 600 cells, 700 cells, 800 cells, 900 cells, 1,000 cells, 1,250 cells, 1,500 cells, 1,750 cells, or 2,000 cells.

[0091] In some embodiments, the composition comprises a cell type derived from an animal, such as a human, rat, or mouse. In some embodiments, the cell type from the animal has one or more characteristics of a disease, such as a neurodegenerative disease, proliferative disease, immune disease, cardiac disease, infectious disease, or metabolic disease. In some embodiments, the composition can be used to study a disease or aspects or factors thereof. In some embodiments, the composition includes HeLa cells, HEK293 cells, DLD-1 cells, U2OS cells, H9C2 cells, induced pluripotent stem cells (iPSCs), cardiomyocytes, myocytes, stem cell-derived cells, neurons, cancer cells, immune cells, or adipocytes. In some embodiments, the cell type is derived from a biopsy or tissue sample, such as from a patient sample, for example, from a healthy or diseased biopsy or tissue sample. In some embodiments, the cell composition is a tissue sample, such as a tissue sample, cell smear, or secretion. When describing a cell type, unless explicitly stated otherwise, it should be understood to include cells derived from that cell type. For example, unless explicitly stated otherwise, HeLa cells containing heterologous transgenes will be considered HeLa cells. In some embodiments, the composition comprises a modified cell type, such as a stable or transiently transfected cell type.

[0092] In some embodiments, the composition is a cultured cell composition, such as a composition contained in sample wells or culture dishes. In some embodiments, the method involves multiple aliquots of the composition, such as aliquots of the composition in two or more wells of a multi-well plate (e.g., a 1536-well plate).

[0093] In other aspects, this document provides methods for identifying, obtaining, and / or preparing compositions comprising cells. In some embodiments, the methods include producing compositions having cell types. In some embodiments, the cell types of the composition are treated and / or engineered to adjust (e.g., increase or decrease) the expression of one or more peptides. Techniques for preparing compositions comprising cells are well known in the art. In some embodiments, cell types are produced from precursor cells of a cell type by modulating some aspect of the precursor. For example, cell types can be generated by subjecting precursor cells to stress (such as oxidative stress) or by treating precursor cells with small molecule compounds or hormones. In some embodiments, cell types are obtained by subjecting precursor cells to infection (such as viral, bacterial, fungal, or parasitic infection). In some embodiments, cell types are generated by knocking down or knocking out a genetic trait or its expression product, such as by any method known in the art, such as siRNA, RNAi, TALEN, ZFN, or CRISPR / Cas. In some embodiments, cell types are generated by knock-in. In some embodiments, cell types are generated by transfection. In some embodiments, cell types are transfected with a fusion peptide (such as a peptide fused to a marker (e.g., GFP)). In some embodiments, cell types are transfected with a wild-type peptide. In some embodiments, cell types are transfected with a variant peptide (such as a mutant peptide). In some embodiments, cell types are transfected to express a predetermined level of gene expression product. In some embodiments, when the gene expression product reaches a predetermined level, a variant cell type with the expression level is generated and used in the methods described herein.

[0094] In some embodiments, the transfected cell type expresses a peptide, such as a wild-type peptide or a mutant peptide (e.g., β-catenin), at near-endogenous levels. In some embodiments, the cell type does not express a peptide, such as a wild-type peptide, and the near-endogenous level is based on the expression level of the peptide in another cell type. In some embodiments, the transfected cell type expresses a labeled peptide, such as a labeled wild-type peptide, at near-endogenous levels, where the near-endogenous level is based on the expression level of the corresponding unlabeled form of the peptide. In some embodiments, the cell type has reduced expression of the unlabeled peptide, for example, the cell type contains a knockout of the unlabeled peptide. In some embodiments, the transfected cell type expresses a variant peptide, such as a mutant peptide (e.g., a point mutation, truncated mutation, frameshift mutation, or termination mutation), whose expression level is substantially similar to the endogenous expression level of the corresponding wild-type peptide of the variant peptide. As described herein, in some embodiments, the terms "near-endogenous level" or "substantially similar" mean that the peptide expression level is within 2-fold difference of the measured endogenous peptide level, such as in a cell population (e.g., 1x10⁻⁶). 6 Measured in individual cells.

[0095] 2. Treatment of compositions containing cells In some aspects, the methods provided herein involve subjecting a cell-containing composition to a stimulus, such as contacting the cell-containing composition with a compound. The methods provided herein can evaluate any stimulus. For ease of description of the subject matter provided herein, compositions are generally described as being subjected to a single stimulus (although this is not a limitation on the description provided herein), and said single stimulus may encompass many factors. For example, the stimulus may be described as a compound, and may also implicitly include any light, heat, O2 concentration, and / or CO2 concentration factors subjected to the cell composition. In some embodiments, the stimulus is described as a concentration of a compound, wherein two or more cell compositions are subjected to different concentrations of the compound (or, for example, a concentration of the compound and a mediator control). Those skilled in the art will readily understand how one or more factors of the stimulus can be evaluated by establishing adequate experimental procedures and references.

[0096] In some embodiments, the stimulus includes an exogenous compound. In some embodiments, the stimulus includes an exogenous peptide, such as a hormone. In some embodiments, the stimulus includes exogenous genetic material. In some embodiments, the stimulus includes a stressor. In some embodiments, the stimulus includes an environmental stimulus. In some embodiments, the stimulus includes any combination of an exogenous compound, an exogenous peptide, exogenous genetic material, a stressor, or an environmental stimulus.

[0097] In some embodiments, the stimulus (such as an exogenous compound) is a small molecule therapeutic agent candidate or its precursor (e.g., a prodrug). In some embodiments, the stimulus is a small molecule therapeutic agent candidate at a known concentration. In some embodiments, the stimulus is a mixture of small molecule therapeutic agent candidates (applied to the cellular composition via one or more compositions). In some embodiments, the stimulus is a small molecule therapeutic agent candidate and another agent (applied to the cellular composition via one or more compositions). In some embodiments, the stimulus is a peptide, peptide mimic, lipid, nucleic acid, or any combination thereof.

[0098] The methods described herein can be used to evaluate any number of stimuli. For example, in the context of drug screening, any number of treatment candidates can be evaluated, such as at least about 1, 2, 5, 10, 50, 100, 1,000, 10,000, 25,000, 50,000, 100,000, 250,000, 500,000, 1,000,000, 2,500,000, or 5,000,000.

[0099] As used herein, small molecule therapeutic candidates encompass any small molecule evaluated for drug discovery purposes, such as efforts to identify therapeutic agents for human diseases. In some embodiments, the small molecule therapeutic agent is a member of a small molecule library (such as a library of chemical compounds). In some embodiments, there may be knowledge (acquired before or after the methods described herein) that a particular small molecule is not suitable for human therapeutic use—in such cases, the small molecule is still considered a small molecule therapeutic candidate for the purposes of this description.

[0100] In some embodiments, the small molecule therapeutic agent candidate has a molecular weight of about 5,000 Da or less, such as about 4,500 Da or less, 4,000 Da or less, 3,500 Da or less, 3,000 Da or less, 2,500 Da or less, 2,000 Da or less, 1,500 Da or less, 1,000 Da or less, or 500 Da or less. In some embodiments, the small molecule therapeutic agent candidate satisfies one or more of the Lipinski five rules—no more than 5 hydrogen bond donors, no more than 10 hydrogen bond acceptors, a molecular weight of less than 500 Da, and an octanol-water partition coefficient (log P) of no more than 5. In some embodiments, the small molecule therapeutic agent candidate has the desired partitioning properties of an aggregate type (e.g., a β-catenin reservoir). For example, in some embodiments, small molecule therapeutic agent candidates are allocated to aggregate types (e.g., the concentration of small molecule therapeutic agent candidates in aggregate types is higher than the concentration outside aggregate types), such as when it is desirable to deliver small molecule therapeutic agent candidates to aggregate types. In some embodiments, small molecule therapeutic agent candidates are substantially not allocated to aggregate types (e.g., the concentration of small molecule therapeutic agent candidates in aggregate types is lower than the concentration outside aggregate types), such as when it is desirable to avoid aggregate types as a library of small molecule therapeutic agent candidates.

[0101] In some embodiments, the small molecule therapeutic agent candidate comprises a nucleic acid. In some embodiments, the small molecule therapeutic agent candidate comprises RNA, such as siRNA, miRNA, mRNA, or lnRNA, or analogs thereof. In some embodiments, the small molecule therapeutic agent candidate comprises DNA or analogs thereof. In some embodiments, the small molecule therapeutic agent candidate is a non-naturally occurring compound. In some embodiments, the small molecule therapeutic agent candidate is an exogenous compound. In some embodiments, the small molecule therapeutic agent candidate comprises a polypeptide. In some embodiments, the small molecule therapeutic agent candidate is a regulatory-approved therapeutic compound, such as one approved for medical use by the U.S. Food and Drug Administration (FDA). In some embodiments, the small molecule therapeutic agent candidate is a novel chemical entity.

[0102] In some embodiments, the small molecule therapeutic agent candidate or a portion thereof is charged. In some embodiments, the small molecule therapeutic agent candidate or a portion thereof is hydrophobic. In some embodiments, the small molecule therapeutic agent candidate or a portion thereof is hydrophilic. In some embodiments, the small molecule therapeutic agent candidate or a portion thereof comprises an alkaloid, glycoside, phenazine, phenol, polyketide, terpene, or tetrapyrrole.

[0103] In some embodiments, the small molecule therapeutic agent candidate comprises a label. In some embodiments, the label is a radioactive label, a colorimetric label, a luminescent label, a chemically reactive label (such as a component portion used in click chemistry), or a fluorescent label. In some embodiments, the small molecule therapeutic agent candidate contains a measurable signal, for example, having fluorescent properties that do not require further modification. In some embodiments, the small molecule therapeutic agent candidate comprises a fluorophore. In some embodiments, the small molecule therapeutic agent candidate is a labeled peptide. In some embodiments, the small molecule therapeutic agent candidate is a peptide containing a fluorophore. In some embodiments, the small molecule therapeutic agent candidate is a labeled nucleic acid. In some embodiments, the small molecule therapeutic agent candidate is a nucleic acid containing a fluorophore. The label may be covalently or non-covalently associated with the small molecule therapeutic agent candidate.

[0104] In some implementations, the stimulus is a reference control, as described herein.

[0105] The methods provided herein are applicable to any therapeutic regimen that includes subjecting the cell composition to stimulation. In some embodiments, subjecting the cell composition to stimulation includes subjecting the cell composition to stimulation in a single instance, such as heat treatment or application of a chemical composition, such as a small molecule therapeutic agent candidate. Such a single treatment may include a duration of any length or incubation, for example, applying a chemical composition for a set time before removing the cell composition culture medium and replacing it with a culture medium that does not contain the chemical composition. In some embodiments, subjecting the cell composition to stimulation includes subjecting the cells to stimulation in multiple instances, such as in response to chronic exposure or chronic disease.

[0106] 3. Measurement method In some aspects, the methods provided herein include independently subjecting any number of cell-containing compositions to stimulation, wherein said methods are capable of evaluating multiple stimuli in one or more cell types. In some embodiments, the methods include repeatedly subjecting the cell composition containing cell types to stimulation. The methods described herein can be implemented in various forms, including those suitable for high-throughput and commercial-scale drug discovery.

[0107] In some embodiments, the methods provided herein include aliquoting a cell-containing composition into a plurality of sample wells and subjecting each defined subset of the aliquoted cell composition to stimulation. In some embodiments, the cell composition is aliquoted into the wells of a multi-well plate, such as a multi-well plate having any of 6, 12, 24, 48, 96, 384, or 1,536 wells. In some embodiments, the multi-well plate is suitable for other steps of the methods described herein, such as stimulation and any processing and analytical steps for the cell type of the cell composition.

[0108] In some embodiments, the methods provided herein include evaluating a variety of cell compositions, each containing a different cell type or a derivative thereof. In some embodiments, each cell composition may be independently subjected to a variety of stimuli, such as by subjecting different aliquots of the cell composition to stimuli.

[0109] In some embodiments, the number of each cell composition used in the methods described herein (including aliquots of cell compositions) may be determined based on one or more aspects of the desired analysis, including but not limited to the number of stimuli to be evaluated, the number of cell types to be evaluated, the number of replicates to be evaluated, and the statistical power level.

[0110] 4. Post-stimulation treatment In some embodiments, the methods provided herein include processing techniques performed after subjecting the cell-containing composition to stimulation. Such processing techniques can be used to preserve the biological background and / or prepare the cell composition or its components for certain downstream analyses after stimulation and prior to such analyses.

[0111] For example, in some embodiments, after stimulating the cell-containing composition, the cell composition or its components (such as cell types) are fixed and / or permeabilized. In some embodiments, fixation is performed to hold the cells in a fixed position, such as using a cross-linking agent (e.g., formaldehyde or an analogue thereof). In some embodiments, permeabilization is performed to allow markers (e.g., antibodies) to penetrate the cells, such as using a solvent (e.g., acetone) and / or a detergent (e.g., Triton, NP-40, Tween 20, saponin, digitonin, and / or leucoperm). In some embodiments, after stimulating the cell composition containing cell types, the cell composition or its components (such as cell types) are frozen.

[0112] D. Additional method steps In some aspects, the methods described herein also include performing one or more orthogonal secondary assays to evaluate compounds identified as regulating β-catenin reservoirs, such as secondary transcription assays examining transcriptional activity of downstream targets of β-catenin. In some embodiments, the methods further include using a second cell-based assay and / or in vitro assay to evaluate the identified compounds. In some embodiments, the methods further include using a second cell-based assay (including, but not limited to, cytotoxicity assays) to evaluate the identified compounds.

[0113] III. The method described in this paper can achieve other aspects. In other respects, this document provides further methods and implementation schemes implemented by the disclosure herein.

[0114] In some aspects, this document provides methods for identifying compounds for treating β-catenin-related diseases, wherein said methods include identifying the compounds according to the methods described herein. In some embodiments, β-catenin-related diseases are cancers.

[0115] In some respects, this article provides compounds identified by the methods described herein.

[0116] In some aspects, this document provides kits and compositions that can be used in the methods described herein. In some embodiments, this document provides kits containing one or more detection reagents, such as antibodies, for measuring (e.g., visualizing) a β-catenin reservoir. In some embodiments, the kits contain one or more cellular compositions that can be used in the methods described herein.

[0117] Those skilled in the art will recognize that several implementations are possible within the scope and spirit of the disclosure of this application. This disclosure is further illustrated by the following examples, which should not be construed as limiting the scope or spirit of this disclosure to the specific procedures described therein.

[0118] VI. Sequence The sequences cited in this article are provided in Table 1.

[0119] Table 1. Sequences.

[0120] Example Example 1 This example demonstrates transient transfection of the fluorophore-β-catenin construct.

[0121] Initial attempts to establish a screening assay for chemical compounds capable of silencing β-catenin transcriptional activity involved transient transfection of cells with a fluorophore-β-catenin construct to visualize β-catenin integration into the nuclear reservoir. Previously, Bernkopf et al. ( Oncotarget (9, 2018) demonstrated that β-catenin labeled with green fluorescent protein transiently transfected into cells could integrate into nuclear spots after treatment with sulforaphane. However, as described below, transient transfection is not robust enough as a technique for compound screening or detection of β-catenin reservoirs.

[0122] Transient transfection and expression of the fluorescent protein-β-catenin construct in HCT116 cells resulted in only a minority of cells exhibiting visible β-catenin nuclear spots. Furthermore, cells found to adequately express this β-catenin construct for imaging typically exhibited high baseline noise and a very small window for detecting potential changes in punctate morphology.

[0123] Transient transfection and expression of the fluorescent protein-β-catenin construct in HEK293T cells resulted in significant changes in cell phenotype, depending on the type of fluorescent label used. The use of these constructs in compound screening is further complicated by the inefficient levels of transfection and the fact that overexpression of these constructs leads to the formation of variable reservoirs dependent on cell cycle stages. Finally, apoptosis was also observed within the time required to screen a large number of compound libraries.

[0124] Example 2 This example demonstrates the detection of β-catenin constructs in a nuclear repository using immunofluorescence.

[0125] Given the numerous challenges associated with transient transfection of β-catenin constructs labeled in the context of compound screening as described in the foregoing embodiments, this study attempts to utilize immunofluorescence to detect β-catenin reservoirs in the cell nucleus and explore the applicability of such detection in compound drug screening.

[0126] U2OS cells were reverse-transfected with a plasmid encoding HaloTag-β-catenin, and then seeded into plates at 2500 cells per well. Cells were treated with sulforaphane or daunorubicin for 6 or 24 hours, followed by HaloTag labeling via exposure to Janelia Fluor® 646. Cells were fixed with 3% (v / v) formaldehyde, washed with phosphate-buffered saline (PBS), permeabilized with 0.2% (v / v) Triton, washed again with PBS, and then blocked with 0.4% (v / v) FSG in PBS.

[0127] After blocking, individual cell wells were exposed to one of several different staining solutions, each containing a different primary antibody targeting a different form of β-catenin. Cells were then washed with PBS and subsequently exposed to another antibody staining solution containing a secondary antibody labeled AlexaFluor® 488, a blocking solution, and 4',6-diamidinyl-2-phenylindole (DAPI) for DNA staining. Cells were washed a final time with PBS.

[0128] This experiment revealed a surprising result: the primary antibody targeting specific phosphorylated epitopes and the biologically inactive form of β-catenin provided a sufficiently high signal-to-noise ratio to accurately measure and count the β-catenin nuclear reservoir. Figure 1 Cells exposed to a primary antibody targeting the inactive form of β-catenin showed prominent nuclear spots co-localizing with HaloTag-β-catenin. However, this was not the case for primary antibodies targeting the active form of β-catenin. Furthermore, β-catenin expressed by the fluorescent protein-β-catenin construct was similarly restricted, as the active form of β-catenin was also tagged and produced a detectable signal.

[0129] Example 3 This example demonstrates label-free detection of β-catenin in nuclear reservoirs and preliminary screening of β-catenin reservoirs.

[0130] Given the surprising findings described in the preceding examples, the next steps were: i) comparing the degree of β-catenin nuclear reservoir formation in wild-type U2OS cells (wt U2OS) and Halo-β-catenin-transfected U2OS cells (Halo U2OS), and ii) performing preliminary screening using a selected set of compounds. In previous experiments, daunorubicin was shown to induce β-catenin nuclear reservoir formation in a similar manner to sulforaphane. Therefore, daunorubicin was selected as a positive control for this assay, and dimethyl sulfoxide (DMSO) was selected as a negative control.

[0131] wt U2OS cells or Halo U2OS cells were seeded into plates at 2500 cells per well. Cells were then treated with different concentrations of daunorubicin. After daunorubicin treatment, Halo U2OS cells were incubated with Janelia Fluor® 646 dye. All cells were then fixed with 3% (v / v) formaldehyde, washed with PBS, permeabilized with 0.2% (v / v) triton, washed again with PBS, and then blocked with 0.4% (v / v) FSG in PBS.

[0132] After blocking, cells were exposed to a staining solution containing a primary antibody against a specific inactive form of β-catenin. Cells were then washed with PBS and exposed to another antibody staining solution containing a secondary antibody labeled Alexa Fluor® 488, blocking solution, and DAPI. Cells were then washed a final time with PBS. Plates were then imaged, and the mean nuclear reservoir size per cell was calculated. The Z-factor was greater than 0.5 for both wt U2OS and Halo U2OS plates.

[0133] The compound dose-response resulted in similar EC50 between wt U2OS and Halo U2OS cells, thus eliminating the need for transient transfection of Halo-β-catenin (and the aforementioned challenges) when screening for compounds that regulate the nuclear reservoir of β-catenin. Figure 2 Exemplary dose-response curves for a single compound in wt U2OS cell assays are depicted.

[0134] Example 4 A larger 20,000-compound phenotypic screening was conducted using label-free detection of the β-catenin reservoir as described in Example 3 above to identify chemicals capable of reversing the overactive function of β-catenin in colorectal cancer by trapping β-catenin in β-catenin reservoir aggregates. U2OS cells were treated with 30 µM of the compound in DMSO for 24 hours. 24 hours after treatment, cells were fixed with formaldehyde and immunofluorescence was performed by incubating the fixed sample with a phosphorylated β-catenin antibody. After washing with PBS, the antigen bound to the antibody was detected using an Alexa Fluor 488-conjugated goat anti-rabbit antibody. The treated cells were imaged using a Phenix high-throughput imaging system, and the levels of phosphorylated β-catenin were quantified using Phenix's accompanying Harmony analysis software. Compounds that significantly enhanced drug reservoir formation compared to DMSO-treated cells were identified as hits and selected for further evaluation and development.

[0135] The identified aggregate modulators (c-mods) were further optimized using traditional structure-activity relationship (SAR) techniques, and their ability to isolate β-catenin into a reservoir, induce selective cancer cell death, and reverse the expression program of oncogenic β-catenin-specific genes was then evaluated.

[0136] As detailed in this article, studies have found that one of the c-mods, compound B, is effective against genetically diverse colorectal cancers and a range of Wnt-related cancers, thus demonstrating its ability to treat a broad patient population. Figure 3-7 ). Figure 3Immunofluorescence (IF) images of healthy and malignant colon cells after treatment with small molecule aggregate regulator B (compound B) are shown. Figure 3 As shown, treatment of malignant cells with compound B resulted in the isolation of β-catenin into aggregates. Figure 4 This study demonstrated the selective killing of cancerous colon cells relative to healthy colon cells after treatment with compound B. Figure 5 Gene set enrichment analysis (GSEA) plots are shown, revealing the selective downregulation of Wnt / β-catenin and LEF1 target genes in colorectal cancer cells when treated with compound B, which is a β-catenin reservoir inducer. Figure 6 The results of treatment with compound B on 99 cancer cell lines are shown. These results indicate that compound B is active in a wide range of Wnt-related cancers.

[0137] Figure 7 Compound B was shown to cause tumor growth arrest in a patient-derived xenograft (PDX) model derived from heavily pretreated CRC patients with high β-catenin expression. FOLFOX is a combination of chemotherapy drugs representing the standard of care for patients with metastatic colorectal cancer. QW = once weekly; BID = twice daily; QD = once daily.

[0138] Furthermore, orally administered c-mod drugs exhibited competitive tumor growth inhibition as a single agent in colorectal cancer xenograft and PDX models (Table 2). In summary, these results highlight the novelty and inventiveness of the method for identifying compounds that regulate β-catenin reservoirs through cellular induction and / or screening. In conclusion, the practicality of compound B described herein demonstrates that the method taught herein for identifying compounds capable of regulating intracellular β-catenin reservoirs represents a significant advance in the fields of drug discovery and the identification of novel therapeutic compositions.

[0139] Table 2. Response of compound B in in vivo models of cancer (including colorectal cancer).

[0140] *CRC = colorectal; LoF = loss of function; GoF = gain of function; wt = wild type; mut = mutation.

Claims

1. A method of identifying a compound that modulates β-catenin pools in a cell, the method comprising: (a) contacting a composition comprising a cell with the compound; (b) determining a β-catenin pool of the composition comprising a cell or a derivative of the composition; and (c) identifying a compound that modulates a β-catenin pool in a cell by comparing the determined β-catenin pool in the cell to a reference.

2. The method of claim 1, wherein comparing the determined β-catenin pool in the cell to the reference is based on one or more of the following attributes of a β-catenin pool: (i) the presence of a β-catenin pool; (ii) the amount of a β-catenin pool; (iii) the size of a β-catenin pool; (iv) the surface area of a β-catenin pool; (v) the cellular localization of a β-catenin pool; (vi) the distribution of a β-catenin pool; (vii) the ratio of a β-catenin pool to the amount of reference condensate; or (viii) the composition of a β-catenin pool.

3. The method of claim 1 or 2, wherein the modulation of a β-catenin pool is based on an increase in the β-catenin pool in the cell compared to the reference.

4. The method of any one of claims 1-3, wherein the modulation of a β-catenin pool is based on an increase in the amount of the β-catenin pool in the cell compared to the reference.

5. The method of any one of claims 1-4, wherein the modulation of the β-catenin pool in the cell is based on an increase in the amount of the β-catenin pool in the nucleus of the cell compared to the reference.

6. The method of any one of claims 1-3, wherein the modulation of a β-catenin pool is based on an increase in the presence of the β-catenin pool in the cell compared to the reference.

7. The method of any one of claims 1-3 and 6, wherein the modulation of a β-catenin pool in the cell is based on an increase in the presence of a β-catenin pool in the nucleus of the cell compared to the reference.

8. The method of any one of claims 1-7, wherein the determination of a β-catenin pool comprises detecting a non-active form of a β-catenin polypeptide.

9. The method of claim 8, wherein detecting a non-active form of a β-catenin polypeptide comprises contacting a composition comprising a cell or a derivative of the composition with a detectable agent that specifically recognizes a non-active form of a β-catenin polypeptide.

10. The method of claim 9, wherein the detectable agent is an antibody that specifically recognizes the non-active form of a β-catenin polypeptide.

11. The method of claim 9 or 10, wherein the determination comprises detecting the detectable agent that specifically recognizes the non-active form of a β-catenin polypeptide.

12. The method of any one of claims 8-11, wherein the detecting comprises imaging at least a portion of the cell. ​ 13. The method of any one of claims 9-12, wherein the detectable agent that specifically recognizes the inactive form of the β-catenin polypeptide is a fluorescently labeled antibody that specifically recognizes the inactive form of the β-catenin polypeptide.

14. The method of any one of claims 9-12, wherein the detectable agent that specifically recognizes the inactive form of the β-catenin polypeptide is detected using a secondary fluorescently labeled antibody that specifically recognizes the detectable agent.

15. The method of any one of claims 9-14, wherein the detectable agent that specifically recognizes the inactive form of the β-catenin polypeptide specifically recognizes phosphorylation at one or more of S33, S37, T41, or S45 of the β-catenin polypeptide, and wherein the amino acid positions are relative to the amino acid sequence of SEQ ID NO:

1.

16. The method of claim 15, wherein the detectable agent that specifically recognizes the inactive form of the β-catenin polypeptide specifically recognizes phosphorylation at T41 and / or S45.

17. The method of claim 16, wherein the detectable agent that specifically recognizes the inactive form of the β-catenin polypeptide specifically recognizes phosphorylation at T41 and S45.

18. The method of claim 17, wherein the detectable agent that specifically recognizes the inactive form of the β-catenin polypeptide is the 23H16L13 antibody clone.

19. The method of any one of claims 1-18, wherein comparing the measured β-catenin reservoir in the cell to the reference is based on one or more of the following attributes of the inactive form of the β-catenin polypeptide associated with the β-catenin reservoir: (i) localization of the inactive form of the β-catenin polypeptide; (ii) amount of the inactive form of the β-catenin polypeptide; (iii) distribution of the inactive form of the β-catenin polypeptide; (iv) aggregate partitioning of the inactive form of the β-catenin polypeptide in the β-catenin reservoir and / or one or more other aggregates; (v) functional activity associated with the inactive form of the β-catenin polypeptide; (vi) aggregation of the inactive form of the β-catenin polypeptide; (vii) post-translational modification state of the inactive form of the β-catenin polypeptide; or (viii) amount of degradation products of the inactive form of the β-catenin polypeptide.

20. The method of claim 19, wherein the modulation of the β-catenin reservoir in the cell is based on an increase in the amount of the inactive form of the β-catenin polypeptide in the β-catenin reservoir compared to the reference.

21. The method of any one of claims 1-20, wherein the measuring of the β-catenin reservoir comprises detecting phosphorylated H2A.X (P-2A.X) and / or DNA damage checkpoint mediator protein 1 (MDC1).

22. The method of any one of claims 1-21, wherein the cell is a model of abnormal Wnt / β-catenin signaling.

23. The method of any one of claims 1-22, wherein the cells are a model of proliferative pathology.

24. The method of any one of claims 1-23, wherein the cell is a model of cancer.

25. The method of any one of claims 1-24, wherein the cell is a cancer cell.

26. The method of claim 25, wherein the cancer cells are human osteosarcoma U2OS cells or human colon adenocarcinoma DLD-1 cells.

27. The method of any one of claims 1-26, wherein the inactive form of the β-catenin polypeptide is a wild-type β-catenin polypeptide.

28. The method of any one of claims 1-26, wherein the inactive form of the β-catenin polypeptide is a mutant β-catenin polypeptide.

29. The method of any one of claims 1-28, wherein the assay further comprises contacting a composition comprising the cells or a derivative thereof with a second detectable reagent, the second detectable reagent specifically recognizing the active form of the β-catenin polypeptide comprising phosphorylated at S552 and / or S675, and wherein the amino acid positions are relative to the amino acid sequence of SEQ ID NO:

1.

30. The method of any one of claims 1-29, further comprising contacting the composition containing the cells or a derivative thereof with a fixative prior to the determination in step (b).

31. The method of any one of claims 1-30, further comprising determining one or more cellular characteristics of the cell.

32. The method of any one of claims 1-31, wherein the reference is based on: (i) A composition comprising cells that have not come into contact with the compound; (ii) A composition comprising cells mixed with a control medium and not in contact with the compound; (iii) Expected β-catenin profile; (iv) the intracellular reference condensate or reference polypeptide; and / or (v) Reference composition containing cells.

33. The method of any one of claims 1-32, further comprising subjecting the composition containing the cells to conditions that promote the formation of a β-catenin reservoir before contacting the composition containing the cells with the compound.

34. The method of any one of claims 1-33, further comprising using a second cell-based assay and / or in vitro assay to evaluate the identified compound.

35. The method of any one of claims 1-34, further comprising using a second cell-based assay to evaluate the identified compound.

36. A method for identifying a β-catenin reservoir in cells, the method comprising: (a) Contacting a cell-containing composition with a detectable reagent that specifically recognizes an inactive form of β-catenin polypeptide phosphorylated at T41 and / or S45; as well as (b) detecting the detectable agent, wherein detecting the detectable agent within the cell identifies a β-catenin reservoir in the cell.

37. The method of claim 36, wherein the detectable agent is an antibody that specifically recognizes a non-active form of the β-catenin polypeptide comprising phosphorylation at T41 and / or S45.

38. The method of claim 37, wherein the detectable agent is an antibody that specifically recognizes a non-active form of the β-catenin polypeptide comprising phosphorylation at T41 and S45.

39. The method of any one of claims 36-38, wherein the detecting comprises imaging at least a portion of the cell.

40. The method of any one of claims 36-39, wherein the detectable agent is a fluorescently labeled antibody that specifically recognizes the non-active form of the β-catenin polypeptide comprising phosphorylation at T41 and / or S45.

41. The method of any one of claims 36-39, wherein the detectable agent is detected using a fluorescently labeled secondary antibody that specifically recognizes the detectable agent.

42. The method of any one of claims 36-41, wherein the method further comprises contacting a composition comprising the cell with a fixative prior to contacting with the detectable agent.

43. The method of any one of claims 36-42, further comprising measuring a signal of the detectable agent.

44. The method of any one of claims 36-43, further comprising comparing the signal to a reference.

45. The method of claim 44, wherein the reference is based on: (i) a composition comprising cells that are not contacted with the detectable agent; (ii) a composition comprising cells mixed with a control vehicle and not contacted with the detectable agent; (iii) a reference signal; (iv) a reference condensate or a reference polypeptide within the cell; and / or (v) a reference composition comprising cells.

46. A method of identifying a compound for treating a β-catenin related disease, wherein the method comprises identifying a compound according to the method of any one of claims 1-35.

47. The method of claim 46, wherein the β-catenin related disease is selected from the group consisting of a cancer, autosomal dominant polycystic kidney disease (ADPKD), hereditary familial adenomatous polyposis (FAP), Alzheimer’s disease, or a metabolic disease.

48. A compound identified by the method of any one of claims 1-35, 46, and 47.

49. A method of identifying a β-catenin reservoir in a cell, the method comprising: (a) contacting a composition comprising a cell with a first detectable agent that specifically recognizes a β-catenin polypeptide; (b) contacting a composition comprising a cell with a second detectable agent that specifically recognizes phosphorylated H2A.X (P-H2A.X) or DNA damage checkpoint mediator protein 1 (MDC1); and (c) detecting the first detectable agent and the second detectable agent. wherein detecting co-localization of the first detectable agent and the second detectable agent within the cell identifies a β-catenin reservoir in the cell.

Images