E7386, a Selective Inhibitor of the Interaction between β-Catenin and CBP, Exerts Antitumor Activity in Tumor Models with Activated Canonical Wnt Signaling

The Wnt/β-catenin signaling pathway (or canonical Wnt signaling pathway) is involved in the control of cellular processes, such as proliferation, differentiation, and motility. Consequently, this pathway is important not only for embryonic development, but also for the development, regulation, and survival of cancer cells (1, 2). Upon the binding of Wnt ligands to Frizzled family receptors on cell membrane, components of the destruction complex, including adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β), and axin, are sequestered to the plasma membrane, where together they prevent degradation of β-catenin. Unphosphorylated β-catenin then translocates to the nucleus, where it forms a complex with the transcriptional factor T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) and its cofactors, such as CREB binding protein (CBP) and p300, leading to target gene transcription.

Genetic alterations, such as activating mutations of the gene encoding β-catenin and loss-of-function mutation of APC, result in aberrant activation of the Wnt/β-catenin signaling pathway (3). These genetic alterations occur in multiple types of cancers, including hepatocellular carcinoma, colorectal cancer, anaplastic thyroid cancer, endometrial cancer, and desmoid tumors (4–9), and so it has been suggested that the activated Wnt/β-catenin signaling pathway is a potential novel therapeutic target. In addition, aberrant activation of the Wnt/β-catenin signaling pathway results in the development of abnormal tumor microenvironments related to tumor immunity, angiogenesis, and metabolism (10–13), and it has been reported that aberrant activation of the Wnt/β-catenin signaling pathway in tumor cells prevents T-cell infiltration into tumors by decreasing the expression of the chemokines necessary for lymphocyte migration (14, 15). Thus, the Wnt/β-catenin signaling pathway is an attractive therapeutic target, not only for direct activity against tumor cells, but also for modulation of the tumor microenvironment to enhance antitumor immunity.

Several Wnt/β-catenin signal inhibitors, including porcupine inhibitors (16), Wnt receptor antibody (17), and Wnt receptor decoy (18), have been developed (19, 20); however, these inhibitors have shown limited antitumor activity and severe toxicity in clinical trials (21, 22). It is difficult to directly target β-catenin itself by using a small molecule-compound inhibitor (23), but some inhibitors, such as ICG-001 and PRI-724, which bind to CBP, inhibit the interaction of CBP with β-catenin, and consequently inhibit the function of β-catenin (24). In clinical trials, the efficacy of PRI-724 administered for 7 consecutive days or biweekly has been examined; however, PRI-724 has not yet been approved for use as anticancer drug. Therefore, novel agents targeting the Wnt/β-catenin signal transcriptional machinery are still needed to improve cancer therapy.

We discovered a novel orally active compound, E7386, that was developed by improving the microsomal stability, membrane permeability, and solubility of C-82, an active form of PRI-724. In this study, E7386 inhibited polyp formation in the intestine in Apc^Min/+ mice, and had antitumor activity in an APC-mutated ECC10 human gastric xenograft model. Mouse mammary tumor virus (MMTV)-Wnt1 transgenic mice develop spontaneous breast tumors with constitutive Wnt1 expression induced by the long terminal repeat region of MMTV. E7386 showed clear antitumor activity when administered alone and synergistic antitumor activity when administered in combination with anti-PD-1 antibody in the MMTV-Wnt1 tumor model. IHC and transcriptome analyses showed that E7386 increased the number of CD8⁺ cells in tumor tissue and altered the immune cell population by decreasing the monocyte and M2 macrophage populations. Together, these findings showed that E7386 had antitumor activity in mouse and human preclinical models by modulating Wnt/β-catenin signaling via inhibition of the β-catenin/CBP interaction.

E7386, ICG-001, and C-82 were synthesized by Eisai Co., Ltd., WuXi AppTec Co., Ltd., and PRISM BioLab Co., Ltd., respectively. For in vitro studies, compounds were prepared as 20 mmol/L stock solutions in DMSO and diluted in the relevant assay medium. For in vivo studies, E7386 was dissolved in 0.1 mol/L HCl, and administration volume was calculated from the body weight of each mouse (0.1 mL/10 g-body weight) before administration.

HEK293 cells were obtained from the ATCC, and ECC10 cells were obtained from RIKEN BioResource Center. Each cell line was cultured according to recommended culture methods and used for no more than 20 passages. ECC10 cells and HEK293 cells were tested negative for Mycoplasma contamination using MycoAlert Mycoplasma Detection Kit (LT07-318, Lonza) and authenticated by short tandem repeat analysis at Promega KK, or Eisai Co., Ltd. by using GenePrint 24 System (Promega), respectively.

FLAG-tagged N-terminal region of CBP (amino acids, 1–682) was transfected into HEK293 cells. The HEK293 transfectants, which were stimulated with 40 mmol/L of lithium chloride (LiCl), and ECC10 cells were treated with the indicated concentration of compounds for 6 hours. The treated HEK293 transfectants and ECC10 cells were lysed with 1 × Cell Lysis Buffer (Cell Signaling Technology) and an EpiQuik Nuclear Extraction Kit II (EpiGentek Group Inc.), respectively, and extracts were incubated with anti-Flag M2 Affinity Gel (Sigma-Aldrich), anti-CBP antibody, or anti-p300 antibody (Santa Cruz Biotechnology) immobilized on agarose overnight at 4°C. Proteins bound to the agaroses were eluted by boiling with SDS sample buffer and then analyzed by Western blotting.

For detection of acetylated β-catenin, HEK293 and ECC10 cells were treated with E7386 for 2 hours. HEK293 cells were also treated with 40 mmol/L of LiCl at the same time to increase acetylation of β-catenin. Treated cells were lysed, whole-cell extracts were separated on 5%–20% SDS–polyacrylamide gradient gels (FUJIFILM Wako Pure Chemical Corporation), and separated proteins were transferred to Nitrocellulose Membranes (GE Healthcare Life Science) by electroblotting. After blocking with TBS containing 5% nonfat dry milk and 0.1% Tween-20, the membranes were incubated individually with the following primary antibodies: acetyl-β-catenin Lys49 (#9030), glyceraldehyde 3-phosphate dehydrogenase antibody (#2118) (Cell Signaling Technology), β-catenin (H-102), CBP (A-22), and p300 (N-15) (Santa Cruz Biotechnology). The membranes were then washed and incubated with secondary horseradish peroxide anti-rabbit IgG antibody. Immunodetection was performed by using Immobilon Western Chemiluminescent HRP Substrate (Millipore) and an LAS-3000 Luminescent Image Analyzer (Fujifilm).

We inserted three copies of the TCF/LEF binding site from TOP Flash Vector (Millipore) into either pNeo luciferase reporter plasmid, which contains the luciferase reporter gene with a mammalian selectable marker for neomycin resistance (constructed by Eisai), or pGL4.19 (Promega), to generate pNeo-TOP and pGL4.19 TOP, respectively. HEK293 cells stably transfected with pNeo-TOP or ECC10 cells transiently transfected with pGL4.19 TOP, and then transfected cells were treated with serial dilutions of E7386 for 6 hours. HEK293 cells were also treated with 40 mmol/L of LiCl at the same time to activate Wnt signaling pathway and reporter activity. TCF/LEF luciferase activity was measured with the Bright-Glo Luciferase Assay System (Promega). TCF/LEF reporter activity was determined as the ratio of luciferase activity with E7386 treatment compared with that without treatment. To determine the inhibitory activity of E7386, we also calculated the ratio of luciferase activity in samples treated with E7386 to that in samples not treated with E7386 (control). IC₅₀ values were calculated by using Prism Software (version 8.0.1, GraphPad Software).

All animal experiments in this study were conducted in accordance with the guidelines for animal experiments of Eisai Co., Ltd. ECC10 cells were injected subcutaneously into the right flank of female nude mice (CAnN.Cg-Foxn1nu/Crl; Charles River Laboratories Japan) and tumors were allowed to grow to 150–200 mm³ in size (designated day 1). MMTV-Wnt1 transgenic mice [B6SJL-Tg(Wnt1)1Hev/J] were obtained from the Jackson Laboratory. Spontaneously developed breast tumors were resected from the MMTV-Wnt1 transgenic mice, diced, and inoculated into C57BL/6J mice (Charles River Laboratories Japan). Tumors were allowed to grow to approximately between 80 and 200 mm³ in size (designated day 1); tumor-bearing mice were then randomly allocated to treatment groups and orally administered with E7386 (12.5, 25, or 50 mg/kg twice daily) or intraperitoneally administered with anti-PD-1 antibody (500 μg/mouse twice weekly). Tumor sizes were measured with digital calipers, and tumor volumes were calculated as long length × short length² × 0.5. Statistical analyses were performed by using Prism software.

Apc^Min/+ mice, which harbor a truncating nonsense mutation at codon 850 of the Apc gene, developed a spontaneous intestinal adenoma (25, 26). Four-week-old Apc^Min/+ mice (C57BL/6J-Apc^Min/J) were orally administered 10 cycles of E7386 (6.25–50 mg/kg twice daily) according to a schedule of 5 days on/2 days off. After treatment, the mice were euthanized, and the polyps in the small intestine were counted under a stereo microscope. Eight-week-old Apc^Min/+ mice were orally administered four cycles of E7386 (30 mg/kg twice daily) according to a same schedule. After the final administration, samples of small intestine and whisker follicles were collected.

Total RNA was extracted from the whisker follicles of Apc^Min/+ mice by using an RNeasy Kit (Qiagen). An aliquot of total RNA (50 ng) was converted to cyanine-3–labeled cRNA by using a Low Input Quick Amp Labeling Kit, One-Color, and then hybridized to a SurePrint G3 Mouse GE 8 × 60K Microarray (Agilent Technologies). Hybridization images were obtained with a DNA Microarray Scanner (Agilent Technologies) and quantified by using Feature Extraction Software (version 11, Agilent Technologies). Raw data were normalized by using the quantile function in R software (version 3.1.0). P values were determined by using Welch t test, followed by adjustment for multiple comparisons by using an FDR approach (Benjamini–Hochberg procedure) in R software (version 3.1.0). A gene was considered differentially expressed when its absolute fold change relative to the control value was ≥2 and it had an FDR P < 0.05. Pathway analysis was conducted by using the Ingenuity Pathway Analysis Platform (Qiagen). The microarray data have been deposited in the NCBI Gene Expression Omnibus under accession number GSE158554.

Total RNA was extracted from ECC10 tumor tissues from mice treated with either vehicle or E7386 (12.5, 25, or 50 mg/kg) and analyzed by using an nCounter Vantage 3D RNA Wnt Pathways Panel (NanoString Technologies). Data were imported into nSolver analysis software (version 4.0) to check the quality, and then normalized on the basis of positive probes and housekeeping genes. Differentially expressed genes (DEG) were identified for each E7386 dose group compared with vehicle by using the following criteria: |fold change| > 1.25 and P < 0.1 (Welch t test). The log₂ ratios of DEGs identified in at least one dose group were visualized as a heatmap by using Strand NGS Software (Agilent Technologies).

Total RNA was extracted from MMTV-Wnt1 tumor tissues resected from mice treated with either vehicle or E7386 at 3 (day 4) and 7 days (day 8) after initiation of each treatment and used for RNA sequencing (RNA-seq) analysis. Sequencing libraries were generated from isolated RNA by using an Illumina TruSeq mRNA Library Kit according to the manufacturer's instructions. Paired-end sequencing of 2 × 150 bp was conducted by using an Illumina NextSeq 500 in accordance with the manufacturer's instructions. FastQ files for each sample converted from the binary base call file were entered into FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) for quality assessment. Sequencing data were processed by using Trimmomatic (version 0.36; ref. 27) to remove all Illumina adaptor sequences and bases with low quality scores. Filtered reads were mapped to the mouse genome (Mm10 and Ensembl 84) by using STAR software (version 2.5.0c; ref. 28). Gene expression levels, in transcripts per million, were estimated for all samples by using RSEM software (version 1.2.31; ref. 29). The RNA-seq data have been deposited in the NCBI Gene Expression Omnibus under accession number GSE158713.

Differential gene expression analysis was performed at Clarivate Analytics by using the DESeq2 R package (30). The most relevant metrics were the fold change in expression relative to the vehicle group, the raw P value derived from the statistical test, and the P value adjusted for multiple comparisons according to Benjamini–Hochberg correction. Topologically significant genes (TS genes) were identified by using four methods: overconnectivity, interconnectivity, network propagation, and causal reasoning (31–35). All of these methods were used for the input; the lists of DEGs were derived from each E7386 dose group compared with vehicle, after splitting genes into those that were downregulated and those that were upregulated. Enrichment analysis using DEGs, TS genes, and the nonredundant union of both gene lists was performed against pathway maps obtained from Metabase at Clarivate Analytics. Cell type deconvolution analysis was performed by using the CIBERSORT algorithm (36), which uses gene expression data to provide an estimation of the abundances of cell types in a mixed cell population.

MMTV-Wnt1 tumor tissues were resected from mice orally administered with 50 mg/kg of E7386 twice daily for a week. Fresh frozen tumor tissues were embedded in Optimal Cutting Temperature Compound (Sakura Finetek) and cut into 10-μm-thick sections. Staining was performed by using anti-CD8 antibody (clone 53-6.7, 1:100, eBioscience) in combination with Alexa Fluor 594–conjugated donkey anti-rat IgG (1:100, Life Technologies), with DAPI (4′,6-diamidino-2-phenylindole, Dojindo) as the counterstain. The sections were scanned by using a White-light Confocal Microscope (Leica Microsystems, TCS SP8), and the CD8⁺ cells within tumors were counted in a blinded manner by using scanned images at 200× magnification.

First, we investigated that E7386 (Fig. 1A) blocked the interaction between the exogenous N-terminal region of CBP and endogenous β-catenin in HEK293 cells overexpressing FLAG-tagged N-terminal region of CBP, and we also examined those reported for ICG-001, the first-reported inhibitor of the β-catenin–CBP interaction (24), and C-82 (Fig. 1A). HEK293 cells were incubated with 40 mmol/L LiCl to inhibit the phosphorylation of β-catenin by GSK3β and activate the Wnt/β-catenin signaling pathway (i.e., induce the interaction between β-catenin and CBP). Coimmunoprecipitation of β-catenin and anti-FLAG antibody from whole-cell lysate was markedly decreased in cells treated at the highest dose for each of the test compounds, indicating that all of the compounds decreased the interaction between β-catenin and CBP (Fig. 1B). On the other hand, coimmunoprecipitation of β-catenin and anti-FLAG antibody was decreased when the lowest dose of E7386 (0.5 μmol/L) was used.

Next, we examined whether E7386 inhibited the interaction of endogenous β-catenin and CBP by using APC-mutated human gastric cancer ECC10 cells, in which the Wnt/β-catenin signaling pathway is highly activated (Supplementary Fig. S1). ECC10 cells were treated with E7386 or ICG-001 for 6 hours, and CBPs in the cell lysate were coimmunoprecipitated with anti-CBP antibody. The amount of β-catenin bound to CBP was determined by Western blotting. E7386 decreased the interaction between β-catenin and CBP in a dose-dependent manner (Fig. 1C). However, neither of the test compounds interfered with the interaction between β-catenin and p300 (Supplementary Fig. S2).

CBP has intrinsic acetyltransferase activity against lysine 49 of β-catenin (37). Therefore, we also examined whether inhibition of the interaction of β-catenin with CBP by E7386 affected acetylation of lysine 49 (Fig. 1D and E). E7386 clearly decreased the acetylation of lysine 49 of β-catenin in both HEK293 and ECC10 cells.

Upon activation of the Wnt/β-catenin signaling pathway, β-catenin enters the nucleus where it engages with DNA-bound TCF transcription factors. Thus, reporter assays using the TCF/LEF binding motif, such as the TOPFlash assay, can be used to examine activation of the Wnt/β-catenin signaling pathway (1). To assess the effect of inhibition of β-catenin and CBP interaction by E7386 on the Wnt/β-catenin signaling pathway, we investigated the effect of E7386 on the Wnt/β-catenin signaling pathway by using a TCF/LEF reporter assay using HEK293 cells stably expressing TCF/LEF luciferase reporter and ECC10 cells transfected with an expression vector for TCF/LEF luciferase reporter (Fig. 1F and G). E7386 treatment for 6 hours inhibited TCF/LEF luciferase activity in a dose-dependent manner in both cell lines. The IC₅₀ value of E7386 in HEK293 and ECC10 cells was 0.0484 and 0.0147 μmol/L, respectively.

We also investigated the effects of ICG-001 and C-82 by using the same TCF/LEF reporter assay (Supplementary Fig. S3A). In HEK293 cells stimulated with LiCl, ICG-001 and C-82 both disrupted TCF/LEF reporter activity in a dose-dependent manner (IC₅₀ values, 3.31 and 0.356 μmol/L, respectively; Supplementary Fig. S3B). Thus, in this assay, the effect of E7386 against the Wnt/β-catenin signaling pathway in HEK293 cells (IC₅₀ value, 0.0484 μmol/L) was stronger than that of ICG-001 and C-82.

Apc^Min/+ mice (C57BL/6J-Apc^Min/J) are genetically engineered with a truncating mutation in the Apc gene, which causes them to spontaneously develop intestinal adenomas due to aberrant activation of the Wnt/β-catenin signaling pathway (25, 26). Apc^Min/+ mice were administered with E7386 (6.25–50 mg/kg orally, twice daily) for 10 cycles (each cycle, 5 days on/2 days off) from 4 weeks of age. After the 10 cycles of treatment, the polyps that formed in the small intestine were counted (Fig. 2A). The number of polyps was 10 times higher in 14-week-old mice than in 4-week-old mice in the vehicle group, and administration of E7386 significantly suppressed polyp formation in a dose-dependent manner compared with the vehicle group (Fig. 2B). Relative body weights on the final day (i.e., ratio of body weight at day 71 to that at day 1) were 1.33 (vehicle), 1.33 (E7386, 6.25 mg/kg), 1.30 (12.5 mg/kg), 1.26 (25 mg/kg), and 1.19 (50 mg/kg). Thus, no marked loss of body weight was observed at any of the doses examined.

The Wnt/β-catenin signaling pathway is reported to be activated in hair and whisker follicles (38, 39). In humans, pre- and posttreatment hair follicles from patients treated with ipafricept, which is a truncated Fzd8 receptor fused to the Fc region of IgG1, showed suppression of Wnt/β-catenin–dependent genes (18). Thus, we next examined whether E7386 disrupted the Wnt/β-catenin signaling pathway by using whisker follicles of Apc^Min/+ mice. E7386 (30 mg/kg orally, twice daily) was administered for four cycles (each cycle, 5 days on/2 days off) from 8 weeks of age. Small intestines and whisker follicles were collected at 6 hours after the final administration. E7386 significantly decreased the number of polyps compared with the vehicle group (Supplementary Fig. S4A). Microarray analysis of total RNA extracted from the whisker follicles of mice treated with E7386 revealed that 183 genes were differentially expressed compared with vehicle treatment (|fold change| ≥ 2; P < 0.05). Subsequent pathway analysis using the ingenuity pathway analysis platform identified WNT3A as the upstream regulator with highest P value of overlap (activation z-score, −2.4; P = 1.77 × 10⁻⁵), indicating that the canonical Wnt/β-catenin signaling pathway was disrupted by E7386 in the whisker follicles of Apc^Min/+ mice (Supplementary Fig. S4B).

E7386 significantly suppressed the growth of ECC10 cells in vitro (IC₅₀ value, 4.9 nmol/L) and induced a cell death in cells exposed to concentrations ≥10 nmol/L for 7 days (Supplementary Fig. S5). This suggested that the proliferation and survival of ECC10 cells are dependent on the Wnt/β-catenin signaling pathway. We, therefore, conducted an in vivo study to examine the antitumor activity of E7386 in the ECC10 tumor xenograft model. ECC10 cells were subcutaneously inoculated into mice, and when the tumor size reached 150 mm³ (designated day 1), the mice were randomly allocated to groups treated with vehicle or E7386 at 12.5, 25, or 50 mg/kg orally, twice daily for 7 days. E7386 showed in vivo antitumor activity in a dose-dependent manner, and significantly inhibited tumor growth when administered at 25 or 50 mg/kg (Fig. 3A) without any significant changes in body weight (Fig. 3B). E7386 treatment for 14 days at 12.5, 25, or 50 mg/kg also showed significant dose-dependent antitumor activity in vivo without any significant changes in body weight (Supplementary Fig. S6A). In contrast, 100 mg/kg of ICG-001 showed only slight antitumor activity and this observation was not statistically significant (Supplementary Fig. S6B).

We then analyzed the expression profile of genes related to the Wnt/β-catenin signaling pathway by using the nCounter analysis platform (Fig. 3C). Gene expression analysis revealed that seven, six, and 41 genes related to the Wnt/β-catenin signaling pathway were differentially expressed in ECC10 tumor tissues resected from mice treated with E7386 at 12.5, 25, or 50 mg/kg, respectively, compared with vehicle treatment (|fold change| > 1.25; P < 0.1; Supplementary Table S1). At 50 mg/kg, E7386 upregulated 19 genes and downregulated 22 genes related to the Wnt/β-catenin signaling pathway. Of the well-known downstream genes of the Wnt/β-catenin signaling pathway, PTGS2 and CCND1 were significantly downregulated and WISP1 was significantly upregulated (Fig. 3C; Supplementary Table S1; refs. 40–42). Collectively, these data suggested that modulation of Wnt/β-catenin signaling pathway by E7386 led to antitumor activity in tumor models showing activated Wnt/β-catenin signaling pathway.

We evaluated the antitumor activity of E7386 in the MMTV-Wnt1 isogenic mouse breast tumor model, in which tumor cells were originated from breast epithelial cells by the Wnt/β-catenin signaling pathway activated by overexpression of Wnt1 under an immunocompetent tumor microenvironment (43). C57BL/6J mice bearing MMTV-Wnt1 tumors, which had been inoculated using diced tumor tissues developed in MMTV-Wnt1 mice, were randomly assigned to treatment groups at 14 days after transplantation, when average tumor sizes reached 220 mm³ (designated day 1), and then E7386 at 12.5–50 mg/kg was orally administered twice daily for 7 days. E7386 significantly inhibited tumor growth in a dose-dependent manner without marked body weight loss (Fig. 4A and B).

Next, we conducted an RNA-seq analysis using total RNA extracted from MMTV-Wnt1 tumor tissues resected from mice orally administered with E7386 at 25 or 50 mg/kg twice daily, or vehicle, for 3 (day 4) or 7 days (day 8) and examined the DEG levels compared with vehicle at days 4 and 8. We observed a higher number of DEGs with P < 0.05 when E7386 was administered at 50 mg/kg (1,363 and 722 at days 4 and 8, respectively) than when it was administered at 25 mg/kg (801 and 501 at days 4 and 8, respectively; Supplementary Table S2). We also identified key regulators, TS genes, of the expression of DEGs by using four methods: overconnectivity, interconnectivity, network propagation, and causal reasoning in the list of DEGs (Supplementary Table S3; refs. 31–35).

We next searched for functional ontologies that were enriched in the list of DEGs and the result of a nonredundant union of both gene lists using pathway maps in Metabase (Supplementary Table S4). The top 10 results of the pathway map analysis at 50 mg/kg on day 8 using the downregulated gene set data revealed that immune-related and hypoxia-related genes were significantly downregulated (Table 1). Among these gene sets, “transcription_HIF-1 targets” and “immune responses_CCL2 signaling” were detected for both the 25 and 50 mg/kg doses with a significant P value. In addition, signaling pathways regulating the tumor microenvironment, including genes related to the innate immune response to contact with allergens or the CD8⁺ Tc1 cell (i.e., cytotoxic T cell) pathway, were significantly upregulated when E7386 was administered at 50 mg/kg (Table 1). These results suggested that E7386 treatment for 7 days altered the tumor microenvironment and immune cell population in tumors.

We next applied the immune cell type deconvolution algorithm CIBERSORT (36) to the RNA-seq data to examine how E7386 affected immune cell populations in tumor tissues (Supplementary Table S5). Twenty-five immune cell subtypes were identified in MMTV-Wnt1 tumor tissues. Among them, monocytes, M2 macrophages, and activated dendritic cells (DC) were decreased, and M0 and M1 macrophages, immature DCs, and CD4⁺ naïve T cells were increased compared with vehicle at day 8 in mice treated with E7386 at 50 mg/kg.

From an IHC analysis, Spranger and colleagues have reported an inverse association between stabilized β-catenin and CD8⁺ T cells in clinical tumor samples (14), suggesting that activation of the Wnt/β-catenin signaling pathway suppressed the infiltration of lymphocytes into tumor. To examine whether disruption of the Wnt/β-catenin signaling pathway by E7386 leads to an increase of CD8⁺ T cells, we further investigated the effect of E7386 on immune activation, CD8⁺ cells in tumor tissue were stained with anti-CD8 antibody and analyzed by fluorescence IHC (Fig. 4C and D); the number of CD8⁺ cells was increased in tumor tissues resected from mice treated with E7386 at 50 mg/kg compared with that in mice treated with vehicle.

β-catenin activation promotes immune escape and resistance to anti-PD-1 therapy (15). To investigate whether the immunomodulatory effect of E7386 is related to its antitumor effect, we tested the antitumor activity of E7386 in combination with anti-PD-1 antibody in a Wnt/β-catenin signaling pathway–activated syngeneic mouse model, the MMTV-Wnt1 mouse breast tumor model. C57BL/6J mice bearing MMTV-Wnt1 tumors were assigned to treatment groups at 14 days after transplantation of MMTV-Wnt1 tumor fragments (designated day 1; when the average tumor size had reached ∼80 mm³), and then treated with E7386 at 50 mg/kg orally, twice daily, anti-PD-1 antibody at 500 μg/mouse intraperitoneally, twice weekly for 21 days, or a combination of both. Anti-PD-1 antibody alone showed no antitumor activity, and E7386 alone showed slow-growing tumors. However, combination treatment of E7386 with anti-PD-1 antibody significantly enhanced the antitumor activity of E7386 (Fig. 5A and B). No severe loss of body weight was observed, indicating that the combination treatment was as tolerable as the monotherapy (Fig. 5C).

Next, we used flow cytometry to investigate the effect of E7386 and combination treatment of E7386 plus anti-PD-1 antibody on the immune cell populations in MMTV-Wnt1 tumors. Immune cell populations were determined by using antibodies listed in Supplementary Table S6, and the gating strategy shown in Supplementary Fig. S7. Among the myeloid populations, both E7386 treatment alone and the combination treatment significantly decreased the M2 macrophage population, gated as CD11b⁺F4/80⁺Ly6C^lowI-A/I-E^lowCD86^low, and significantly increased the M1/M2 macrophage ratio (Supplementary Fig. S8A–S8D). The CD8⁺ T-cell population tended to be increased by combination treatment compared with vehicle, although this change was not statistically significant. However, the CD8⁺ T-cell/CD4⁺ T-cell ratio was significantly increased by both anti-PD-1 antibody treatment alone and the combination treatment compared with vehicle (Supplementary Fig. S8E–S8H). Consistent with our CIBERSORT findings, CD8⁺ T cells in MMTV-Wnt1 tumor were upregulated by combination treatment accompanied by a decrease of M2 macrophages and increase of the M1/M2 ratio.

Collectively, these data indicated that E7386 showed synergistic combination antitumor activity with anti-PD-1 antibody in the MMTV-Wnt1 model.

Here, we found that E7386 inhibited the interaction between β-catenin and CBP in vitro and that orally administered E7386 inhibited the development of adenoma polyps in Apc^Min/+ mice. In vivo, E7386 inhibited the growth of ECC10 human xenografted tumors in nude mice, as well as the growth of MMTV-Wnt1 mouse breast tumors in immunocompetent mice with induction of tumor-infiltrating lymphocyte. Moreover, E7386 exhibited synergistic antitumor activity in combination with anti-PD-1 antibody in a MMTV-Wnt1 mouse tumor model.

The effect of E7386 against the Wnt/β-catenin signaling pathway was more potent than the activities of ICG-001 and C-82, which are previously reported inhibitors of the β-catenin/CBP interaction (Fig. 1; Supplementary Fig. S3). Oral administration of E7386 at 6.25 mg/kg suppressed intestinal polyp formation in Apc^Min/+ mice, whereas PRI-724 at 300 mg/kg was required to achieve comparable inhibition (Supplementary Fig. S9). Because E7386 has 80-fold metabolic stability compared with C-82 (Supplementary Table S7), it may contribute to lower doses of E7386 to show inhibitory activity in addition to the intrinsic potent activity of E7386 compared with PRI-724.

In vitro, E7386 inhibited the growth of ECC10 cells at concentrations near the Wnt/β-catenin signaling pathway (Fig. 1G; Supplementary Fig. S5). These results suggest that the proliferation of ECC10 cells is dependent on the Wnt/β-catenin pathway and that E7386 inhibits cell growth by modulating Wnt/β-catenin signaling. The clear antitumor activity of E7386 in the ECC10 xenograft model (Fig. 3) and the findings of the DEGs analysis of ECC10 tumors were consistent with this hypothesis. In the DEGs analysis of genes related to the Wnt/β-catenin signaling pathway in the ECC10 tumor xenograft model, E7386 was found to decrease the expression of CCND1 and increase the expression of WISP1 (Supplementary Table S1). It has been reported that CBP occupies the CCND1 promoter and that ICG-001 inhibits the occupancy of this site by CBP (24). WISP1 is reported to be a β-catenin/p300 target gene (44). These previous reports suggest that E7386-mediated modulation of Wnt/β-catenin downstream genes in ECC10 tumor cells results in selective inhibition of the interaction between β-catenin and CBP. Emami and colleagues have shown that ICG-001, previously reported analogue compound of E7386, binds to amino acids 1–111 of CBP, and that the complex between these amino acids and β-catenin is inhibited by treatment with ICG-001 (24). In contrast, they have also shown that ICG-001 does not inhibit the binding of β-catenin to amino acids 1 through 111 of p300. Together with our data showing that E7386 blocks the interaction between β-catenin and CBP, but not between β-catenin and p300, these findings suggest that the activity of E7386 is likely due to an increase in the binding of β-catenin with p300.

In our RNA-seq analysis using the MMTV-Wnt1 mouse breast tumor model, signaling pathways related to immunity and inflammation, such as “immune response_CCL2 signaling,” were downregulated at day 8 in animals administered with E7386 at 25 and 50 mg/kg. In addition, a signaling pathway related to hypoxia was significantly downregulated at days 4 and 8 when E7386 was administered at 25 mg/kg (Table 1; Supplementary Table S4). It has been reported that CCL2 and hypoxia signals play important roles in immune resistance and suppression of immune status by tumor microenvironments (45, 46). From our results, it was hypothesized that E7386 altered the immunosuppressive tumor microenvironment by modulation of the Wnt/β-catenin signaling pathway.

Consistent with the findings of our DEGs analysis, immune deconvolution analysis revealed that E7386 altered immune cell populations by increasing the immunostimulatory macrophage population (i.e., M1 macrophages) and decreasing the immunosuppressive macrophage population (i.e., M2 macrophages), resulting in an increase in the M1/M2 macrophage ratio. It has been reported that activation of the Wnt/β-catenin signaling pathway promotes M2 macrophage polarization, and that knockdown of Wnt ligand inhibits tumor growth in association with a decrease in the M2 macrophage population and an increase in the cytotoxic T lymphocyte population (47). In addition, CCL2 is a key chemokine for polarizing macrophages toward M2 macrophages (48). In this study, we found that E7386 increased the number of CD8⁺ cells in MMTV-Wnt1 tumor tissue (Fig. 4C). Together, these findings suggest that E7386-mediated modulation of the Wnt/β-catenin signaling pathway decreases M2 macrophages and increases tumor-infiltrating CD8⁺ cells.

Lymphocyte infiltration and an IFNγ signature define the T-cell–inflamed tumor phenotype (referred to as “hot tumor”), and their presence is essential for anti-PD-1 antibody therapy to be effective. In contrast, “cold tumors” lack lymphocyte infiltration and an IFNγ signature and are resistant to anti-PD-1 antibody therapy (49, 50). Activation of the Wnt/β-catenin signaling pathway in the tumor microenvironment causes T-cell exclusion. Wnt/β-catenin signal–activated murine melanoma shows impaired priming of antitumor T cells and suppression of the recruitment of CD103⁺ DCs because of decreased expression of CCL4 (14). Consistent with the roles of the Wnt/β-catenin pathway in those previous reports, in this study, we found that the MMTV-Wnt1 mouse breast tumor model was resistant to anti-PD-1 antibody and that the number of CD8⁺ cells was low in MMTV-Wnt1 tumor tissue (Figs. 4C and 5A and B). However, E7386 showed synergistic antitumor activity in combination with anti-PD-1 antibody compared with each monotherapy treatment in the MMTV-Wnt1 mouse breast tumor model, where activated Wnt/β-catenin pathway might cause cold tumors. In fact, E7386 decreased the M2 macrophage population and increased lymphocyte infiltration into MMTV-Wnt1 tumor tissue. Although additional studies are needed to understand the precise immunomodulatory mechanism of E7386, we hypothesize that E7386 may alter the immunosuppressive tumor microenvironment to enhance the antitumor activity of anti-PD-1 antibody.

In conclusion, we demonstrated that E7386 exerted antitumor activity, an immunomodulatory effect, and synergistic antitumor activity in combination with anti-PD-1 antibody via inhibition of the interaction of β-catenin with CBP in human and in mouse preclinical tumor models with activation of the Wnt/β-catenin signaling pathway. On the basis of these preclinical results, E7386 monotherapy is currently being investigated in phase I clinical trials for patients with solid tumors in the United Kingdom and Japan (trial identifiers NCT03264664 and NCT03833700).

K. Yamada reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. S. Inoue reports a patent for WO2015098853 issued and US20150175615 issued. Y. Yamamoto reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work, as well as has a patent for US2015175615 issued to Eisai Co., Ltd., and WO201598853 issued to Eisai Co., Ltd. K. Iso reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work, as well as has a patent for WO2015098853 issued and US20150175615 issued. H. Kamiyama reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. A. Yamaguchi reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. T. Kimura reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. M. Uesugi reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. M. Matsuki reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work, as well as has a patent for WO2018147275A1 pending. K. Nakamoto reports personal fees from Eisai. Co., Ltd., during the conduct of the study and outside the submitted work. H. Harada reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. N. Yoneda reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. A. Takemura reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. I. Kushida reports personal fees from Eisai. Co., Ltd., during the conduct of the study and outside the submitted work. N. Wakayama reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. Y. Kato reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. T. Semba reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. A. Yokoi reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. M. Iwata reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. T. Uenaka reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. J. Matsui reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. T. Matsushima reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. T. Owa reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. Y. Funahashi reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work. Y. Ozawa reports personal fees from Eisai Co., Ltd., during the conduct of the study and outside the submitted work, as well as has a patent for WO2016/204193 pending. No disclosures were reported by the other authors.

K. Yamada: Validation, investigation, visualization, methodology, writing-original draft. Y. Hori: Validation, investigation, visualization, methodology. S. Inoue: Design and synthesis of E7386. Y. Yamamoto: Design and synthesis of E7386. K. Iso: Design and synthesis of E7386. H. Kamiyama: Validation, investigation, visualization, methodology. A. Yamaguchi: Validation, investigation, visualization, methodology. T. Kimura: Validation, investigation, visualization, methodology. M. Uesugi: Investigation, visualization, methodology. J. Ito: Formal analysis. M. Matsuki: Methodology. K. Nakamoto: Design and synthesis of E7386. H. Harada: Design and synthesis of E7386. N. Yoneda: Design and synthesis of E7386. A. Takemura: Design and synthesis of E7386. I. Kushida: Validation, investigation, methodology. N. Wakayama: Validation, investigation, methodology. K. Kubara: Methodology. Y. Kato: Validation, investigation, visualization, methodology, writing-review and editing. T. Semba: Conceptualization, project administration. A. Yokoi: Conceptualization, project administration. M. Matsukura: Conceptualization, project administration, design and synthesis of E7386. T. Odagami: Supervision. M. Iwata: Supervision. A. Tsuruoka: Supervision. T. Uenaka: Supervision. J. Matsui: Supervision. T. Matsushima: Supervision. K. Nomoto: Supervision. H. Kouji: Supervision. T. Owa: Supervision. Y. Funahashi: Supervision, writing-review and editing. Y. Ozawa: Conceptualization, validation, investigation, visualization, methodology, project administration, writing-review and editing.

We gratefully acknowledge Shuntaro Tsukamoto and Saori Ikeda (Eisai Co., Ltd.) and Sunplanet Co., Ltd., for their technical support with the experiments.

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