Compositions and methods for mitigating epithelial barrier dysfunction

Abstract

The invention provides methods for treating a disease or disorder characterized by epithelial barrier dysfunction by modulating CARD14:MYC interaction, and related methods and compositions for modulating MYC activity in target epithelial cells, the methods comprising contacting the epithelial cells with an inhibitor or an agonist of CARD14 binding to MYC in the target epithelial cells.

Description

COMPOSITIONS AND METHODS FOR MITIGATING EPITHELIAL BARRIER DYSFUNCTIONSTATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

[0001] This invention was made with Government support under contract AI070235 awarded by the National Institutes of Health. The Government has certain rights in the invention.BACKGROUND

[0002] New therapeutic targets and methods are needed for the treatment of diseases and disorders associated with dysfunctional epithelial barriers. The present invention addresses this need.BRIEF SUMMARY

[0003] The present invention is based, in part, on the discovery of an interaction between CARD 14 and MYC in epithelial cells which modulates the activity of MYC and, in the disease context, leads to epithelial barrier dysfunction manifesting in a disease or disorder, as described herein. Accordingly, the present invention provides methods and related compositions for restoring healthy epithelial barrier function by modulating the interaction between CARD 14 and MYC.

[0004] In one aspect, provided is a method for modulating MYC activity in target epithelial cells, the method including contacting the epithelial cells with an inhibitor or an agonist of CARD14 binding to MYC in the target epithelial cells.

[0005] The method may also include where the epithelial cells are contacted with an inhibitor of CARD 14 binding to MYC, thereby activating MYC activity in the target epithelial cells.

[0006] The method may also include where the epithelial cells are contacted with an agonist of CARD14 binding to MYC, thereby inhibiting MYC activity in the target epithelial cells.

[0007] The method may also include where the target epithelial cells are in vitro, in vivo, or ex vivo.

[0008] The method may also include where the target epithelial cells are within skin or airway epithelial barrier tissues.

[0009] The method may also include where the target epithelial cells are skin, nasal, tracheal, lung, intestinal, prostate, breast, cervical, or vaginal epithelial cells, optionally where the skin epithelial cells are keratinocytes.

[0010] The method may also include where the inhibitor binds to CARD14.

[0011] The method may also include where the agonist binds to CARD 14.

[0012] In one aspect, provided is a method for treating a disease or disorder characterized by epithelial barrier dysfunction in a subject in need thereof, the method including modulating MYC activity in target epithelial cells by administering an inhibitor or an agonist of CARD 14 binding to MYC in the target epithelial cells.

[0013] The method may include activating MYC activity in the target epithelial cells by administering an inhibitor of CARD 14 binding to MYC in the target epithelial cells.

[0014] The method may include inhibiting MYC activity in the target epithelial cells by administering an agonist of CARD 14 binding to MYC in the target epithelial cells.

[0015] The method may also include where the disease or disorder is selected from the group consisting of asthma, atopic dermatitis, allergic rhinitis, celiac disease, chronic rhinosinusitis, eosinophilic esophagitis, food allergy, inflammatory bowel disease, and psoriasis.

[0016] The method may also include where the disease or disorder is atopic dermatitis.

[0017] The method may also include where the disease or disorder is asthma, allergic rhinitis, or chronic rhinosinusitis.

[0018] The method may also include where the disease or disorder is a food allergy or eosinophilic esophagitis.

[0019] The method may also include where the disease or disorder is characterized by reduced MYC activity in the target epithelial cells.

[0020] The method may also include where the disease or disorder is psoriasis.

[0021] The method may also include where the disease or disorder is characterized by increased MYC activity in the target epithelial cells.

[0022] The method may also include where the agonist or inhibitor is an anti-CARD14 antibody, or a peptide or polypeptide CARD14-binding fragment thereof.

[0023] The method may also include where the agonist or inhibitor is a human or humanized anti-CARD14 monoclonal antibody, or a peptide or polypeptide CARD14-binding fragment thereof.

[0024] The method may also include where the agonist or inhibitor is a single or double stranded RNA interference-based agent (RNAi) targeted to inhibit the expression of the CARD 14 gene.

[0025] The method may also include where the agonist or inhibitor is selected from a microRNA, a short hairpin RNA, or a short interfering RNA ("siRNA").

[0026] The method may also include where the CARD14 agonist or inhibitor is a small molecule, a peptide, a polypeptide, or an antibody that binds to CARD14.

[0027] The method may also include where MYC activity is determined relative to MYC activity in the absence of the CARD14 agonist or inhibitor.

[0028] The method may also include where the subject is a human subject.

[0029] The method may also include where the inhibitor or agonist of CARD14 binding to MYC is administered orally, intranasally, by inhalation, topically, or intradermally.

[0030] The method may also include where MYC activity is determined by a method including assessing an amount of nuclear localization of MYC in the target epithelial cells in the presence and absence of the CARD14 agonist or inhibitor, where an increase in the amount of nuclear MYC indicates activation of MYC activity and a decrease in the amount of nuclear MYC indicates inhibition of MYC activity in the target epithelial cells.

[0031] The method may also include where MYC activity is determined by a method including assessing MYC transcriptional activity in the target epithelial cells in the presence and absence of the CARD14 agonist or inhibitor, where an increase in MYC transcriptional activity indicates activation of MYC activity and a decrease in MYC transcriptional activity indicates inhibition of MYC activity in the target epithelial cells.

[0032] In one aspect, provided is a CARD 14 binding agent, where the CARD 14 binding agent inhibits binding of CARD14 to MYC.

[0033] The CARD14 binding agent may also include where the agent is a small molecule, a peptide, a polypeptide, or an antibody.

[0034] In one aspect, provided is a CARD14 binding agent, where the CARD14 binding agent is an agonist of CARD 14 binding to MYC.

[0035] The CARD14 binding agent may also include where the agent is a small molecule, a peptide, a polypeptide, or an antibody.

[0036] In one aspect, provided is a pharmaceutical composition including a CARD14 binding agent, where the composition is formulated as an oral dosage form, an injectable dosage form, a topical dosage form, an intratracheal dosage form, or an inhalable dosage form.

[0037] Other technical features may be readily apparent to one skilled in the art from the following figures, descriptions, and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0038] FIG. 1A illustrates a schematic of the differential expression experiment.

[0039] FIG. IB is a volcano plot illustrating differential expression between wild-type HaCaT human keratinocytes and HaCaT human keratinocytes with the R820W variant.

[0040] FIG. 1C illustrates results from gene set enrichment analysis (GSEA).

[0041] FIG. ID shows a heatmap of GSEA MYC target gene expression in each treatment condition.

[0042] FIG. IE illustrates the results of an unbiased IPA network analysis for HaCaT human keratinocytes carrying the R820W variant, which indicates MYC as a central regulator.

[0043] FIG. 2A is a plot showing relative expression of total MYC protein in four conditions as assessed by Western analysis. Expression is relative to the housekeeping gene, beta-actin

[0044] FIG. 2B is a plot showing fold change in nuclear localization of MYC in the four conditions. Expression is relative to the nuclear housekeeping gene, TATA-binding protein.

[0045] FIG. 2C shows immunofluorescent staining for nuclear MYC in wild-type cells (974) or cells homozygous for the R820W variant (811), each either untreated (NT) or treated with mepazine (M).

[0046] FIG. 2D illustrates the autophagic degradation process, including where the autophagy inhibitor bafilomycin Al acts.

[0047] FIG. 2E is a plot showing relative MYC autophagic flux (ratio in bafilomycin Al- treated vs. vehicle-treated cells) in wild-type HaCaT human keratinocytes (WT) or HaCaT human keratinocytes homozygous for the R820W variant (R820W) in the presence or absence of mepazine (M).

[0048] FIG. 3A is a plot showing proliferation in wild-type HaCaT human keratinocytes (WT) or HaCaT human keratinocytes homozygous for the R820W variant (R820W), each either untreated or treated with mepazine (M).

[0049] FIG. 3B is a plot showing percentage of cells positive for Ki67 in wild-type HaCaT human keratinocytes (WT) or HaCaT human keratinocytes homozygous for the R820W variant (R820W), each either untreated or treated with mepazine (M).

[0050] FIG. 3C shows representative images from immunohistochemical analyses of Ki67 expression in engineered skin substitutes generated from homozygous wild-type (WT) or homozygous R820W variant (R820W) primary human keratinocytes and primary human fibroblasts, each treated with either vehicle or mepazine.

[0051] FIG. 3D shows representative histological images depicting epidermal thickness in engineered skin substitutes generated from from homozygous wild-type (WT) or homozygous R820W variant (R820W) primary human keratinocytes and primary human fibroblasts, each treated with either vehicle or mepazine.

[0052] FIG. 3E is a plot showing quantification of full epidermal thickness, including SC, in engineered skin substitutes generated from wild-type primary human keratinocytes and primary human fibroblasts (WT, strain 974, dark circles) or primary human keratinocytes and primary human fibroblasts homozygous for the R820W variant (R820W, strain 811, open circles), each treated with either vehicle (NT) or mepazine (M).

[0053] FIG. 3F is a plot showing quantification of stratum corneum thickness, in engineered skin substitutes generated from wild-type primary human keratinocytes and primary human fibroblasts (WT, strain 974, dark circles) or primary human keratinocytes and primary human fibroblasts homozygous for the R820W variant (R820W, strain 811, open circles), each treated with either vehicle (NT) or mepazine (M).

[0054] FIG. 3G is a plot showing quantification of epidermal thickness, without SC, in engineered skin substitutes generated from wild-type primary human keratinocytes and primary human fibroblasts (WT, strain 974, dark circles) or primary human keratinocytes and primary human fibroblasts homozygous for the R820W variant (R820W, strain 811, open circles), each treated with either vehicle (NT) or mepazine (M).

[0055] FIG. 3H is a schematic showing the air-liquid interface (ALI) model utilized to measure transepidermal / endothelial electrical resistance (TEER).

[0056] FIG. 31 depicts three graphs showing, from left to right, Baseline TEER between WT and R820W primary human keratinocytes; TEER in homozygous WT primary keratinocytes; and TEER in homozygous R820W primary keratinocytes.

[0057] FIG. 4A illustrates results of an immunoprecipitation experiment with HA-tagged MYC and FLAG-tagged CARD 14.

[0058] FIG. 4B shows representative images from proximity ligation assays (PLA) conducted in engineered skin substitutes (ESS) and normal human skin of both homozygous wild-type and R820W CARD14 genotypes. Images from ESS are shown. Similar images are observed in normal human skin.

[0059] FIG. 4C illustrates results of an immunoprecipitation experiment with HA-tagged MYC and FLAG-tagged CARD 14, including PMA / ionomycin stimulation and mepazine treatment groups.

[0060] FIG. 4D illustrates predicted binding interaction between the bHLH domain of MYC and the MAGUK domain of CARD14. The binding site for the small molecule MYC inhibitor 10058-F4 on the MYC molecule is also indicated.

[0061] FIG. 4E illustrates results of an immunoprecipitation experiment with HA-tagged MYC and FLAG-tagged CARD14, including treatment with the small molecule MYC inhibitor, 10058-F4.

[0062] FIG. 5A shows results from gene set enrichment analysis (GSEA).

[0063] FIG. 5B shows results from gene set enrichment analysis (GSEA).

[0064] FIG. 5C shows results from gene set enrichment analysis (GSEA).

[0065] FIG. 5D shows results from gene set enrichment analysis (GSEA).

[0066] FIG. 5E shows results from gene set enrichment analysis (GSEA).

[0067] FIG. 5F shows results from gene set enrichment analysis (GSEA).

[0068] FIG. 6 is a schematic representation of the relationship between NFKB and MYC signaling in CARD14-associated inflammatory skin diseases.

[0069] FIG. 7 is a schematic representation showing a model of how the R820W variant can predispose both AD and psoriasis by modulating MYC activity.

[0070] FIG. 8 illustrates a model of how R820W impacts keratinocyte MYC activity.DETAILED DESCRIPTION

[0071] As used herein, the term “MYC” refers to cellular myc protein, or “c-myc”. The term “MYC activity” refers to MYC signal transduction activity which may be measured by methods known in the art. For example, MYC signal transduction activity can be measured usingtranscriptional reporter assays. MYC signal transduction activity can also be measured by assays that detect changes in the subcellular location of the MYC protein under specified conditions, for example, changes in cytoplasmic and nuclear levels of the MYC protein. MYC signal transduction activity can also be measured by assessing changes in one or more transcription signatures of recognized MYC regulated genes, including MYC “hallmark” signatures as determined by prior GSE analysis. In accordance with the methods described here, MYC activity may be measured in at least two conditions, such as two or more treatment conditions, or MYC activity may be measured in reference to a control, reference, or standard condition, such as a healthy condition or cells carrying a reference polymorphism.

[0072] As used herein, the term “CARD14” refers to the CARD14 / CARMA2 protein product of the CARD 14 / C ARMA2 gene, unless context indicates the gene is being referred to.CARD 14 is a member of the caspase recruitment domain (CARD)-containing membrane- associated guanylate kinase family of scaffold proteins. The CARD14 / CARMA2 protein is expressed in keratinocytes of the skin, as well as within the epithelia of several additional tissues, including the lungs, esophagus, vagina, cervix, and small intestine according to publically-available Genome Tissue Expression (GTEx) data. Mutations in the CARD14 / CARMA2 gene have been associated with chronic inflammatory skin disorders and some isoforms have been indicated as strong inducers of NF-kB signaling.

[0073] A common single nucleotide polymorphism (SNP) in the human CARD14 / CARMA2 gene, identified as rsl 1652075 (C2458T) (also referred to as exml363660), produces an arginine (R) to tryptophan (W) missense variant in the CARD14 protein (R820W) and has been associated with psoriasis in humans. The R820W protein variant is associated with reduced expression of the barrier gene FLG, as described previously in DeVore, S. B. et al. Novel Role for Caspase Recruitment Domain Family Member 14 and its Genetic Variant rsl 1652075 in Skin Filaggrin Homeostasis. J. Allergy Clin. Immunol. S0091-6749(21)01100-3 (2021).

[0074] As used herein, the term “R820W” refers to the R820W protein variant associated with the SNP rsl 1652075.

[0075] The term “agonist" in the context of “an agonist of CARD14 binding to MYC” refers to an agent that increases or stabilizes CARD 14 binding to MYC. In embodiments, the agonist binds to CARD14. In embodiments, the agonist may be an antibody, or a peptide or polypeptide CARD14-binding fragment thereof. In embodiments, the agonist may be a small moleculeagonist. In embodiments, the agonist may be a polysaccharide. In embodiments, the agonist may be a polynucleotide, including for example an RNA molecule.

[0076] The term “inhibitor" in the context of “an inhibitor of CARD 14 binding to MYC” refers to an agent that blocks, decreases or destabilizes CARD 14 binding to MYC. In embodiments, the inhibitor binds to CARD 14. In embodiments, the inhibitor may be an antibody, or a peptide or polypeptide CARD14-binding fragment thereof. In embodiments, the inhibitor may be a small molecule inhibitor. In embodiments, the inhibitor may be a polysaccharide. In embodiments, the inhibitor may be a polynucleotide, including for example an RNA molecule. In some embodiments, the inhibitor is a CARD 14 binding agent that prevents a new binding interaction between CARD14 and MYC. In some embodiments, the agent is a CARD14 binding agent that disrupts an existing binding interaction between CARD 14 and MYC.

[0077] The term “small molecule” refers to a small organic molecules having a low molecular weight, typically less than or equal to about 1000 daltons, or about 900 daltons.

[0078] The following discussion describes experiments demonstrating an interaction between CARD 14 and MYC in epithelial cells which modulates the activity of MYC. Also discussed is how, in the disease context, inappropriate inhibition of MYC signaling by CARD14 leads to epithelial barrier dysfunction.

[0079] FIG. 1A to FIG. IE show that inhibition of the CARD14 variant, R820W, or inhibition of the CARD 14 signaling mediator, MALT1, by mepazine, reduces MYC transcriptional activity in keratinocytes.

[0080] FIG. 2A to FIG. 2E show that reduced cellular and nuclear MYC in R820W and mepazine-treated cells is attributable to increased MYC autophagy.

[0081] FIG. 3A to FIG. 31 show that proliferation and barrier function is attenuated in R820W and mepazine-treated keratinocytes.

[0082] FIG. 4A to FIG. 4E show that CARD 14 and MYC directly interact in keratinocytes, that this interaction is lost upon CARD14 activation in a genotype-dependent manner, and that the interaction can be disrupted by a small molecule inhibitor of MYC that binds to the bHLH domain of MYC.

[0083] FIG. 5 A to FIG. 7 show that altered MYC signatures are detectable in the skin of individuals with the rsl 1652075 variant and inflammatory skin diseases and provide proposedmodels for the relationship between NFKB and MYC signaling in CARD14-associated inflammatory skin diseases and how the R820W variant can predispose both AD and psoriasis by modulating MYC activity.

[0084] Wild-type HaCaT human keratinocytes were CRISPR-edited to generate a HaCaT cell line homozygous for the rsl 1652075 C>T exonic variant giving rise to the R820W protein variant but otherwise isogenic with wild-type (WT) HaCaT human keratinocytes. In the following discussion, the terms “R820W cells” or “R820W variant” are used interchangeably to refer to the HaCaT human keratinocytes carrying the rsl 1652075 C>T variant.

[0085] First, differences in global RNA expression between WT and R820W variant human HaCaT keratinocytes were analyzed. Both cell lines were treated with either vehicle or mepazine, an inhibitor of the CARD 14 signaling mediator MALT1. RNA from the four conditions was subjected to RNA-sequencing and analyzed for differential expression analysis using the DESeq2 package in R.

[0086] FIG. 1A shows a schematic of the RNA-sequencing experiment between vehicle- and mepazine treated WT and R820W HaCaT human keratinocytes.

[0087] FIG. IB shows a volcano plot of the 1,752 differentially-expressed genes (FDR <0.01) in HaCaT human keratinocytes carrying the R820W variant compared to wild-type (WT) HaCaT human keratinocytes.

[0088] FIG. 1C shows results from gene set enrichment analysis (GSEA) which reveals significant enrichment for downregulated hallmark MYC target genes in HaCaT human keratinocytes carrying the R820W variant. Reduced MYC signatures are also observed with mepazine treatment in both wild-type cells and cells carrying the R820W variant (data not shown). These data indicate there is reduced MYC transcriptional activity in R820W and mepazine-treated HaCaT human keratinocytes.

[0089] FIG. ID shows a heatmap of the expression of GSEA-defined hallmark MYC target genes in each treatment condition as determined by RNA-sequencing, from left, wild-type (WT) no treatment (NT); WT mepazine treatment (M); R820W NT; and R820W M. The heatmap indicates attenuated MYC target gene expression in HaCaT human keratinocytes carrying the R820W variant and in wild-type cells treated with mepazine compared to untreated wild-type cells.

[0090] Tables 1 and 2 show the results of Ingenuity Pathway Analysis (IP A) performed on differentially-expressed genes (DEGs; FDR <0.01) between WT and R820W HaCaT humankeratinocytes. For HaCaT human keratinocytes carrying the R820W variant, IPA analysis predicted attenuated MYC activity and dysregulated molecular and cellular functions classically associated with MYC activity. The same prediction was observed for mepazine treated cells (data not shown).

[0091] Table 1: IPA Upstream Regulators: R820W vs WT HaCaT KeratinocytesPredicted Activation Z-Name P-value Activation scoreP-estradiol 9.43* 10’71Inhibited -2.583Tretinoin 7.01 * 10'51Activated 6.475MYC 2.09 * 1049Inhibited -5.910TGFB1 6.33* IO’48Inhibited -2.456TP53 7.27* 10’48N / A 0.720

[0092] Table 2: IPA Molecular & Cellular Functions: R820W vs WT HaCaT Keratinocytes#Name P-value RangeMolecules4.20*10'13toCellular Movement 5965.72* 1O’601.51*1013toCell Death and Survival 7521.07*10'562.45* 10'13toCellular Development 7861.40*10'54Cellular Growth and 2.45*1 O’13to751 Proliferation 1.40*1 O'54Cellular Assembly and 2.98*10'13to406 Organization 1.48*1 O’42

[0093] FIG. IE shows the results of an unbiased IPA network analysis on the DEGs (FDR <0.01) between WT an R820W HaCaT human keratinocytes. The analysis indicates that inhibited MYC (circled) activity is predicted to be a central hub.

[0094] HaCaT human keratinocytes were treated with mepazine or vehicle and total MYC protein was determined by Western blot analysis. FIG. 2A shows reduced levels of total MYC protein in cells carrying the R820W variant and in keratinocytes of both genotypes treated with mepazine. Different symbols represent different replicates.

[0095] In the same experimental paradigm, nuclear localization of MYC protein was also examined. FIG. 2B shows reduced levels of nuclear MYC protein in cells carrying the R820W variant and in keratinocytes of both genotypes treated with mepazine. Different symbols represent different replicates.

[0096] Three-dimensional human engineered skin substitutes (ESS) were generated from patient-matched primary human keratinocytes and primary human fibroblasts derived from homozygous WT or homozygous R820W individuals and treated with or without mepazine. Resulting ESS sections were subjected to immunofluorescent staining for MYC. FIG. 2C shows results of quantification of the MYC intensity within the nuclei (which were delineated by the presence of the nuclear stain DAPI). The results indicate reduced nuclear MYC in R820W and mepazine-treated ESS. Arrows indicate the location of select nuclei in the paired images for each condition. Closed circles represent homozygous WT ESS measurements; open circles represent homozygous R820W ESS measurements.

[0097] A diagram of the autophagic degradation process is shown in FIG. 2D. The diagram indicates where in the process the small molecule bafilomycin Al exerts its effect to halt autophagy completion and allow the accumulation of autophagic target proteins. “Autophagic flux” is defined as the ratio of the amount of target protein with bafilomycin Al treatment compared to vehicle treatment.

[0098] HaCaT human keratinocytes were treated with bafilomycin Al or vehicle and MYC protein was determined by Western analysis. As shown in FIG. 2E, relative to WT, there was increased autophagic flux of MYC protein in R820W human HaCaT keratinocytes and mepazine- treated keratinocytes. These data indicate greater degradation of MYC by autophagy in R820W and mepazine-treated HaCaT keratinocytes, thus indicating that the reduced MYC protein in R820W and mepazine-treated HaCat keratinocytes is attributable to autophagic degradation. Each replicate is represented by a different symbol.

[0099] In FIG. 3A, cell proliferation is determined using a flow cytometry-based cell trace violet assay. As shown in the figure, the proliferation rate in cells carrying the R820W variant and in mepazine-treated HaCaT human keratinocytes is significantly reduced compared to WT and vehicle-treated cells. Each replicate is represented by a different symbol.

[0100] Another useful indicator of cell proliferation is the nuclear antigen Ki67 which is a marker of active cell proliferation. FIG. 3B shows a reduced frequency of cells positive for high Ki67 expression in HaCaT human keratinocytes carrying the R820W variant and in keratinocytes treated with mepazine.

[0101] Ki67 expression was also evaluated by immunohistochemical analysis in three- dimensional human engineered skin substitutes (ESS) generated from patient-matched primary human keratinocytes and primary human fibroblasts derived from homozygous WT or homozygous R820W individuals and treated with or without mepazine. FIG. 3C shows a clear reduction in Ki67 positive cells in ESS generated from primary cells carrying the R820W and / or treated with mepazine, which is suggestive of reduced proliferation.

[0102] There was also an overall reduction in epidermal thickness in an ESS generated from homozygous R820W vs homozygous WT primary human keratinocytes and primary human fibroblasts, as shown in FIG. 3D.

[0103] As shown in FIG. 3E, FIG. 3F, and FIG. 3G, the reduction in epidermal thickness was driven by reduced thickness in the proliferating cell layers (i.e., strata basale, spinosum and granulosum) rather than changes in the stratum corneum (SC). Closed circles represent homozygous WT measurements from strain 974; open circles represent homozygous R820W measurements from strain 811. These data further support a proliferative defect in human primary keratinocytes harboring the R820W variant.

[0104] FIG. 3H is a schematic showing the air-liquid interface (ALI) model utilized to measure transepidermal / endothelial electrical resistance (TEER), a measure of barrier function.For this assay, primary human keratinocytes were seeded onto transwells and differentiated for 14 days, during which TEER readings were regularly taken.

[0105] FIG. 31 shows results of TEER experiments. Maximum TEER is lower in primary human keratinocytes homozygous for the R820W variant (n=3) compared to homozygous WT keratinocytes (n=3), indicating a baseline reduction in barrier function in cells carrying the R820W variant. Mepazine and the MYC inhibitor 10058-F4 further attenuate maximum TEER in primary keratinocytes of either genotype, highlighting the importance of MALT 1 in this mechanism and the crucial role of MYC activity in formation of a functional epidermal barrier, which are the functional readouts of keratinocyte differentiation. The crucial role of MYC in the epidermal barrier formation and function has been as previously reviewed by Watt, F. M., Frye, M. & Benitah, S. A. MYC in mammalian epidermis: how can an oncogene stimulate differentiation? Nat. Rev. Cancer 8, 234-242 (2008).

[0106] To investigate whether CARD14 directly binds to MYC, HA-tagged MYC and FLAG- tagged CARD14WTor FLAG-tagged CARD14R820Wwere co-transfected into CARD 14-defici ent (KO) HaCaT keratinocytes. FIG. 4A shows HA-tagged MYC and both FLAG-tagged CARD14 isoforms co-immunoprecipitated with each other. These data support the existence of an interaction between CARD14 and MYC.

[0107] To investigate whether endogenous CARD 14 directly binds to endogenous MYC in human tissue, proximity ligation assays (PLA) were conducted in engineered skin substitutes (ESS) and normal human skin of both homozygous wild-type and R820W CARD14 genotypes. FIG. 4B shows the presence of positive PLA signals in ESS of both genotypes. Select nuclei are indicated with an “N”, and select PLA puncta (i.e., locations of CARD14-MYC interaction within the ESS) are indicated by arrows. These data indicate that CARD14 and MYC directly interact within the epidermal keratinocytes of ESS regardless of CARD14 genotype. Positive PLA signals are also detectable in the normal human skin of both genotypes, further indicating the presence of a direct interaction between CARD14 and MYC in human skin tissue. In addition, PLA signals in untreated HaCaT human keratinocytes transfected with HA- and mCherry-tagged MYC (HA-HA-MYC-mCherry) and FLAG- and eGFP-taggedCARD14WT(CARD14WT-FLAG-eGFP) indicate a baseline interaction between MYC and CARD14. Upon stimulation of CARD14 signaling with PMA and ionomycin, cytoplasmic PLA puncta diminished while nuclear HA-HA-MYC-mCherry intensity increased, indicating cytoplasmic CARD14 activation promotes MYC dissociation and nuclear localization. Manilset al. eLife 9, e56720 (2020) generated mice with inducible expression of the gain-of-function missense mutation E138A (Cardl4E138A)' . GSEA analysis of RNA-seq data from Cardl4EI38Avs WT mice identified enrichment of upregulated hallmark MYC target genes. This is consistent with our data suggesting that CARD14 activation induces MYC activity.

[0108] As shown in FIG. 4C, stimulation of CARD14 signaling with PMA and ionomycin attenuates MYC co-immunoprecipitation with CARD14WT, but not CARD14R820W, in transfected HaCaT cells, suggesting that R820W may reduce MYC activity, in part, by preventing its nuclear localization. There was no significant difference in GSEA enrichment of hallmark MYC targets in KO vs WT HaCaT human keratinocytes. However, KO keratinocytes have upregulated MYC signatures compared to R820W HaCaT human keratinocytes, further suggesting CARD14R820Wis defective in promoting MYC activity compared to CARD14WT, which can readily do so.

[0109] In silica modeling identified a strong potential binding interaction between MYC’s bHLH domain and the CARD14 protein’s MAGUK domain, depicted graphically in FIG. 4D. Notably, R820W is oppositely located from this predicted binding site, which may explain why both WT and R820W forms of CARD 14 are able to interact with MYC at baseline.

[0110] In order to further probe the direct interaction between MYC and CARD 14, cells were treated with the small molecule MYC inhibitor, 10058-F4, which binds MYC’s bHLH domain as shown in FIG. 4D and thus would be expected to inhibit MYC binding to CARD14 based on the in silico modeling described above. As shown in FIG. 4E, MYC and CARD 14 failed to co- immunoprecipitate after a 24 hour treatment with 10058-F4. These data indicate that the interaction between MYC and CARD14 can be successfully disrupted by the presence of a small molecule such as 10058-F4.[OHl] Next, gene sets from three studies, Li, B. et al. Transcriptome analysis of psoriasis in a large case-control sample: RNA-seq provides insights into disease mechanisms. J. Invest. Dermatol. 134, 1828-1838 (2014); Liu, J. et al. Transcriptomic Profiling of Plaque Psoriasis and Cutaneous T-Cell Subsets during Treatment with Secukinumab. JID Innov. Skin Sci. Mol. PopuL Health 2, 100094 (2022); and Jordan, C. T. et al. PSORS2 is due to variants in CARD14. Am. J. Hum. Genet. 90, 784-795 (2012) were utilized to assess the expression of hallmark MYC target genes in the skin of individuals with the rsl 1652075 variant and inflammatory skin diseases. FIG. 5A shows reduced MYC gene expression signatures with theR820W variant in healthy skin. The R820W variant in psoriatic skin also trended towards reduced MYC gene expression signatures, as shown in FIG. 5B.

[0112] Psoriatic skin, which is known to have elevated CARD14 activity, had increased MYC expression signatures compared to health skin by GSE analysis, as shown in FIG. 5C . The GSE dataset GSE54456 was used for healthy skin. These data support the model that elevated CARD 14 activity enhances MYC signaling in keratinocytes.

[0113] The anti-IL-17A antibody, secukinumab, which is an established treatment for psoriasis, prevents activation of the IL-17A receptor (IL-17R). IL-17R is known to activate CARD14 signaling as shown previously by DeVore, S. B. et al. Novel Role for Caspase Recruitment Domain Family Member 14 and its Genetic Variant rsl 1652075 in Skin Filaggrin Homeostasis. J. Allergy / Clin. Immunol. S0091-6749(21)01100-3 (2021); and Wang, M. et al. Gain-of-Function variant of Cardl4 Leads to Spontaneous Psoriasis-like Skin Inflammation through Enhanced Keratinocyte Response to IL-17A. Immunity 49, 66-79. e5 (2018).

[0114] FIG. 5D shows that secukinumab is also associated with reduced MYC activity in psoriatic skin. These data support the model that reduced CARD 14 activity attenuates MYC signaling in keratinocytes.

[0115] The G117S and E138A CARD14 mutations cause elevated CARD14 signaling as defined by elevated NFKB signaling, with the effect of E138A being greater than G117S. CARD14 G117S and E138A are associated with psoriasis vulgaris (PsV) and the more severe inflammatory disease generalized pustular psoriasis (GPP). FIG. 5E shows that elevated MYC expression signatures are also present in the skin of individuals with the G117S and E138A CARD14 mutations. MYC signatures are greater in the skin of individuals with E138A than those with G117S. These data further support the model that increased CARD14 activity enhances MYC signaling in human keratinocytes.

[0116] Gene sets from two studies, Greuter, T. et al. Characterization of eosinophilic esophagitis variants by clinical, histological, and molecular analyses: A cross-sectional multicenter study. Allergy 77, 2520-2533 (2022), and Kasela, S. et al. Genetic and non-genetic factors affecting the expression of COVID- 19-relevant genes in the large airway epithelium. Genome Med. 13, 66 (2021) were utilized to assess the expression of hallmark MYC genes in the bronchial and esophageal tissue of individuals with the inflammatory skin diseases allergic asthma and eosinophilic esophagitis. As shown in FIG. 5F, GSEA analysis of these genes sets show significantly attenuated MYC activity in the context of these inflammatory diseasescompared to healthy bronchial and esophageal tissue, respectively. Given the expression of CARD 14 in bronchial and epithelial tissue, these data indicate that an interaction between CARD 14 and MYC may be therapeutically targeted in other epithelia in the context of other inflammatory diseases. GSE datasets were from individuals with Th2-high asthma (GSE67472) or eosinophilic esophagitis (EoE) (GSE148381).

[0117] FIG. 6 is a schematic representation of the relationship between NFKB and MYC signaling in CARD14-associated inflammatory skin diseases.

[0118] FIG. 7 is a schematic representation showing a model of how the R820W variant can predispose both AD and psoriasis by modulating MYC activity.

[0119] FIG. 8 illustrates a model of how R820W impacts keratinocyte MYC activity, and how inhibiting CARD14-MYC interaction can be utilized to promote healthy epidermal / epithelial barrier development and function, particularly in inflammatory epithelial diseases that are associated with reduced MYC activity.

Claims

CLAIMSWhat is claimed is:

1. A method for modulating MYC activity in target epithelial cells, the method comprising contacting the epithelial cells with an inhibitor or an agonist of CARD14 binding to MYC in the target epithelial cells.

2. The method of claim 1, wherein the epithelial cells are contacted with an inhibitor of CARD 14 binding to MYC, thereby activating MYC activity in the target epithelial cells.

3. The method of claim 2, wherein the inhibitor binds to CARD14.

4. The method of claim 1, wherein the epithelial cells are contacted with an agonist of CARD14 binding to MYC, thereby inhibiting MYC activity in the target epithelial cells.

5. The method of claim 4, wherein the agonist binds to CARD14.

6. The method of any one of claims 1 to 5, wherein the target epithelial cells are in vitro, in vivo, or ex vivo.

7. The method of any one of claims 1 to 5, wherein the target epithelial cells are within skin or airway epithelial barrier tissues.

8. The method of any one of claims 1 to 7, wherein the target epithelial cells are skin, nasal, tracheal, lung, intestinal, prostate, breast, cervical, or vaginal epithelial cells, optionally wherein the skin epithelial cells are keratinocytes.

9. A method for treating a disease or disorder characterized by epithelial barrier dysfunction in a subject in need thereof, the method comprising modulating MYC activity in target epithelial cells by administering an inhibitor or an agonist of CARD14 binding to MYC in the target epithelial cells.

10. The method of claim 9, wherein the method comprises activating MYC activity in the target epithelial cells by administering an inhibitor of CARD14 binding to MYC in the target epithelial cells.

11. The method of claim 9, wherein the method comprises inhibiting MYC activity in the target epithelial cells by administering an agonist of CARD14 binding to MYC in the target epithelial cells.

12. The method of any one of claims 9 to 11, wherein the disease or disorder is selected from the group consisting of asthma, atopic dermatitis, allergic rhinitis, celiac disease, chronic rhinosinusitis, eosinophilic esophagitis, food allergy, inflammatory bowel disease, and psoriasis.

13. The method of claim 10, wherein the disease or disorder is atopic dermatitis.

14. The method of claim 10, wherein the disease or disorder is asthma, allergic rhinitis, or chronic rhinosinusitis.

15. The method of claim 10, wherein the disease or disorder is a food allergy or eosinophilic esophagitis.

16. The method of claim 10, wherein the disease or disorder is characterized by reduced MYC activity in the target epithelial cells.

17. The method of claim 11, wherein the disease or disorder is psoriasis.

18. The method of claim 11, wherein the disease or disorder is characterized by increased MYC activity in the target epithelial cells.

19. The method of any one of claims 1-14 or 16-18, wherein the CARD14 agonist or inhibitor is a small molecule, a peptide, a polypeptide, or an antibody that binds to CARD 14.

20. The method of claim 19, wherein the agonist or inhibitor is an anti-CARD14 antibody, or a peptide or polypeptide CARD14-binding fragment thereof.

21. The method of claim 20, wherein the agonist or inhibitor is a human or humanized anti- CARD14 monoclonal antibody, or a peptide or polypeptide CARD14-binding fragment thereof.

22. The method of claim 1, wherein the agonist or inhibitor is a single or double stranded RNA interference-based agent (RNAi) targeted to inhibit the expression of the CARD 14 gene.

23. The method of claim 22, wherein the agonist or inhibitor is selected from a microRNA, a short hairpin RNA, or a short interfering RNA ("siRNA").

24. The method of any one of claims 1 to 23, wherein MYC activity is determined relative to MYC activity in the absence of the CARD14 agonist or inhibitor.

25. The method of claim 24, wherein MYC activity is determined by a method comprising assessing an amount of nuclear localization of MYC in the target epithelial cells in the presence and absence of the CARD14 agonist or inhibitor, wherein an increase in the amount of nuclear MYC indicates activation of MYC activity and a decrease in the amount of nuclear MYC indicates inhibition of MYC activity in the target epithelial cells.

26. The method of claim 24, wherein MYC activity is determined by a method comprising assessing MYC transcriptional activity in the target epithelial cells in the presence and absence of the CARD 14 agonist or inhibitor, wherein an increase in MYC transcriptional activity indicates activation of MYC activity and a decrease in MYC transcriptional activity indicates inhibition of MYC activity in the target epithelial cells.

27. The method of any one of claims 9-14 or 16-26, wherein the subject is a human subject.

28. The method of any one of claims 9-14 or 16-27, wherein the inhibitor or an agonist of CARD14 binding to MYC is administered orally, intranasally, by inhalation, topically, or intradermally.

29. A CARD 14 binding agent, wherein the CARD 14 binding agent inhibits binding of CARD 14 to MYC.

30. A CARD14 binding agent, wherein the CARD14 binding agent is an agonist of CARD14 binding to MYC.

31. The CARD 14 binding agent of claim 29 or 30, wherein the agent is a small molecule, a peptide, a polypeptide, or an antibody.

32. A pharmaceutical composition comprising a CARD 14 binding agent, wherein the composition is formulated as an oral dosage form, an injectable dosage form, a topical dosage form, an intratracheal dosage form, or an inhalable dosage form.

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