WNTinib is a multi-kinase inhibitor with specificity against β-catenin mutant hepatocellular carcinoma

Abstract

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. β-Catenin (CTNNB1)-mutated HCC represents 30% of cases of the disease with no precision therapeutics available. Using chemical libraries derived from clinical multi-kinase inhibitor (KI) scaffolds, we screened HCC organoids to identify WNTinib, a KI with exquisite selectivity in CTNNB1-mutated human and murine models, including patient samples. Multiomic and target engagement analyses, combined with rescue experiments and in vitro and in vivo efficacy studies, revealed that WNTinib is superior to clinical KIs and inhibits KIT/mitogen-activated protein kinase (MAPK) signaling at multiple nodes. Moreover, we demonstrate that reduced engagement on BRAF and p38α kinases by WNTinib relative to several multi-KIs is necessary to avoid compensatory feedback signaling-providing a durable and selective transcriptional repression of mutant β-catenin/Wnt targets through nuclear translocation of the EZH2 transcriptional repressor. Our studies uncover a previously unknown mechanism to harness the KIT/MAPK/EZH2 pathway to potently and selectively antagonize CTNNB1-mutant HCC with an unprecedented wide therapeutic index.

Extended Data Fig. 1 |. Chemical genetic screens in tumor organoids yield WNTinib as a selective antagonist of CTNNB1-mutated HCC.

a, The base structures of sorafenib and regorafenib were used as starting points for kinase inhibitor development. b, The required perfluoroalkyl-substituted aniline building blocks were obtained in a single step from aniline. c, In cases where isocyanates were not commercially available, the required acyl imidazole intermediates were generated in situ from an aniline and N,N’-carbonyldiimidazole. d, In the final inhibitor generating step, the urea linker component was formed via reaction of a commercially available isocyanate or in situ formed acyl imidazole with a core aniline corresponding to sorafenib (X = H) or regorafenib (X = F). e, Key interactions and predicted binding pose of sorafenib and regorafenib analogs. Points of diversification are highlighted as X and R, which are specified for each analog in panel d. f-i, IC₅₀ curves of WNTinib (f), 8–50-2 (g), sorafenib (h), and regorafenib (i) in murine HCC organoids used in Fig. 1a. N = 3 independent experiments [mean, SEM]. j. Caspase activity in CTNNB1-mutant models treated with WNTinib at 1 μM for 3 days. N = 3 independent experiments [mean, SEM]. Extended data associated with Fig. 1.

Extended Data Fig. 2 |. WNTinib reduces EZH2 phosphorylation to drive the suppression of essential gene networks in CTNNB1-mutated HCC.

a, Principal component analysis (PCA) of phosphoproteomics displaying MYC-CTNNB1 and MYC-Tp53 tumor organoids treated or not with WNTinib for 4 or 24 hours, as related to Fig. 2a. b. Volcano plots depicting phosphoproteomic changes elicited by WNTinib in MYC-CTNNB1 tumor organoids (left) or MYC-Tp53 tumor organoids (right) as compared to DMSO. Inset: pathway enrichment terms associated with significantly regulated phosphoproteins. WNTinib was used at 1 μM for 4 hours. N = 2 independent experiments. Gene-centered, differentially expressed phosphosites were determined by using estimates of variance-mean dependence with a Benjamini-Hochberg FDR correction. c-d, Interaction networks and pathway enrichment for the significantly up and downregulated phosphoproteins in the MYC-CTNNB1 (c) and MYC-Tp53 (d) models treated with WNTinib used at 1 μM for 24 hours. Strength of interactions denoted by STRING P value. Proteins driving pathway enrichment shown in boxes. e-f, Clustered heatmap of the combined score for kinase-substrate predictions (a higher combined score indicates a stronger kinase-substrate prediction) for the top substrates (y-axis) and kinases (x-axis) modulated by WNTinib in MYC-CTNNB1 (e) and MYC-Tp53 (f) tumor organoids. Pathway enrichment for both kinases and substrates was done using STRING. Substrates driving enrichment shown in boxes; EZH2 highlighted in red. Extended data associated with Fig. 2.

Extended Data Fig. 3 |. WNTinib reduces EZH2 phosphorylation to drive the suppression of essential gene networks in CTNNB1-mutated HCC.

a-b, PCA of transcriptomics (a) displaying MYC-CTNNB1 tumor organoids treated or not with WNTinib for 1 hour, 24 hours, or 7 days, as related to Fig. 2b. N = 2 independent experiments. b, Volcano plots depicting transcriptomic changes elicited by WNTinib as related to panel a. Inset: pathway enrichment terms associated with significantly regulated transcripts. c-d, PCA of transcriptomics (c) displaying MYC-Tp53 tumor organoids treated or not with WNTinib for 1 hour, 24 hours, or 7 days, as related to Fig. 2b. N = 2 independent experiments. d, Volcano plots depicting transcriptomic changes elicited by WNTinib as related to panel c. Inset: pathway enrichment terms associated with significantly regulated transcripts. For panels b, d – differential events were identified using two-sided Wald tests with Benjamini-Hochberg multiple testing corrections. Extended data associated with Fig. 2.

Extended Data Fig. 4 |. WNTinib reduces EZH2 phosphorylation to drive the suppression of essential gene networks in CTNNB1-mutated HCC.

a, Heat maps and average profile plots for MYC-CTNNB1 tumor organoids (right) or MYC-Tp53 tumor organoids (left) displaying CUT&RUN H3K27me3 signal around the TSS/TES ( + /− 3 kb) of genes unchanged by WNTinib in the MYC-CTNNB1 tumor organoids (RNAseq – 7 days; N = 3767). Genes were chosen for similar expression levels to WNTinib-modulated genes. b, As in a, but displaying H3K27me3 levels at genes belonging to the Multicellular Organism Development Pathway in WNTinib-treated MYC-CTNNB1 tumor organoids. c, Representative genome browser tracks of H3K27me3 levels in WNTinib-treated MYC-CTNNB1 tumor organoids. Top: examples belong to Wnt Signaling Pathway; Bottom: example of a housekeeping gene. d, ChIP-qPCR enrichment of H3K27me3 at the promoters of genes shown in panel c. MYC-CTNNB1 tumor organoids were treated with DMSO, sorafenib (10 μM), or WNTinib (1 μM) for the indicated times. N = 3 independent experiments [mean, SEM]. P values; Sox7 *.00698 (1 day), *.0085 (3 day), *.01045 (7 day); Wnt11 *.02699 (1 day), *.02624 (3 day), *.02432 (7 day); Wnt7a *.01741 (1 day), *.01263 (3 day), ***.00045 (7 day); as calculated with two-tailed paired t-tests. Extended data associated with Fig. 2.

Extended Data Fig. 5 |. WNTinib depends on EZH2 relocalization to chromatin for its activity in CTNNB1-mutated HCC.

a, Schematic of phospho-site regulation of EZH2 by WNTinib in the MYC-CTNNB1 tumor organoids. Predicted kinases for each site shown in boxes. b, Western blot depicting the modulation of pT367 EZH2 by WNTinib (1 μM) or sorafenib (10 μM) in the four tumor organoid models used in Fig. 1a. Tumor organoids were treated for 24 hours. c-d, Cytoplasmic and nuclear fractions of total and pT367 EZH2 from MYC-CTNNB1 organoids (c) or HEPG2 cells (d) treated with DMSO, sorafenib (10 μM organoids, 5 μM cells), or WNTinib (1 μM organoids, .5 μM cells) for 24 hours. Tubulin and histone H3 used as fractionation controls. e, IC₅₀ curves for WNTinib (left) or sorafenib (right) in MYC-CTNNB1 tumor organoids depleted for EZH2 (with two independent shRNA targeting EZH2). Inset: western blot depicting depletion efficiency. N = 3 independent experiments [mean, SEM]. f, Western blot depicting total EZH2 degradation as related to Fig. 3g. g, WNT reporter expression levels in MYC-CTNNB1 organoids treated with WNTinib (1 μM), GSK343 (1 μM), or MS1943 (1 μM) alone or in combination. Values obtained from three biological replicates [mean, SEM, n = 3]. Significant differences between groups indicated by asterisks. * P < .05, ** P < .005, as calculated with two-tailed, paired t-tests. h, RNA expression levels of genes in MYC-CTNNB1 tumor organoids depleted for EZH2 and treated with DMSO, sorafenib (10 μM), or WNTinib (1 μM). Genes are classified as being described PRC2 targets or not. N = 3 independent experiments [mean, SEM]. Significant differences between groups indicated by asterisks. * P < .05, ** P < .005, *** P < .0005 as calculated with two-tailed, paired t-tests. Exact P values listed in source data. Western blot results were independently validated at least two times. Extended data associated with Fig. 3.

Extended Data Fig. 6 |. WNTinib utilizes unique polypharmacology to regulate the EZH2-WNT axis.

a, Kinome selectivity for sorafenib, regorafenib, 8–50-2, and WNTinib. Y-axis indicates the number of kinases that each compound inhibits at >65%, as profiled using KINOMEscan. b-e, Trees depicting the kinome inhibition profiles of WNTinib (b), 8–50-2 (c), sorafenib (d), and regorafenib (e). f, Time course (1 to 24 hours) of signaling perturbations on pT367 EZH2. HEPG2 cells were treated with DMSO, sorafenib (5 μM) or WNTinib (.5 μM). Western blot measures endogenous proteins as indicated. g, Supernatant transfer experiment in HEPG2 cells. Cells were first treated with DMSO, sorafenib (5 μM), WNTinib (.5 μM), or vemurafenib (10 μM) for 48 hours. Supernatants were harvested and applied to fresh cells for the indicated times and pT367 EZH2 levels were measured. h, IC₅₀ curves and i, Heatmap depicting IC₅₀ values associated with each treatment condition in Fig. 5d–e. HEPG2 cells were transduced with constructs encoding constitutively active MEK (S218D, S222D) or MKK6 (S207E, T211E). Parental HEPG2 cells used as control comparison. Cells were treated for 3 days. Values obtained from three biological replicates [mean, SEM, n = 3]. j, WNT reporter expression levels in HEPG2 transduced as in panel h and treated or not with WNTinib (.5 μM) for 24 hours. Parental HEPG2 cells used as control comparison. N = 3 independent experiments [mean, SEM]. Significant differences between groups indicated by asterisks. * P < .05, as calculated with two-tailed paired t-tests. P values; *.0291 (WNTinib), *.0181 (MEK WNTinib), *.0352 (MKK6 WNTinib). Western blot results were independently validated at least two times. Extended data associated with Figs. 4 and 5.

Extended Data Fig. 7 |. KIT is a critical target for WNTinib’s MoA.

a, IC₅₀ curves and b, Heatmap depicting IC₅₀ values associated with each treatment condition in Fig. 6e–f. HEPG2 cells were transduced with a doxycycline (DOX)-inducible construct encoding constitutively active cKIT (V559D, T670I). Cells not treated with DOX used as control comparison. Cells were treated for 3 days. Values obtained from three biological replicates [mean, SEM, n = 3]. c, Growth curves for MYC-CTNNB1 tumor organoids (left) or MYC-Tp53 tumor organoids (right) depleted for KIT using shRNA. Depletion efficiency represented in panel d. N = 3 independent experiments [mean, SEM]. Significant differences between curves (as compared to untreated) indicated by asterisks. *** P < .0005, as calculated with a two-way ANOVA with Tukey test for multiple comparisons (F (4, 20) = 38.99). d, pT367 EZH2 and H3K27me3 modulation in shKIT MYC-CTNNB1 tumor organoids and MYC-Tp53 tumor organoids. Western blot measures endogenous proteins as indicated. e, IC₅₀ curves for WNTinib in MYC-CTNNB1 tumor organoids depleted for KIT. Organoids were treated for 3 days. N = 3 independent experiments [mean, SEM]. For panel c, P values; ***<.0001 (shKit-1, shKit-2). Extended data associated with Fig. 6.

Extended Data Fig. 8 |. BRAF and p38 kinases are critical anti-targets for WNTinib.

a, IC₅₀ curves and b, Heatmap depicting IC₅₀ values associated with each treatment condition in Fig. 7a–b. HEPG2 cells were transduced with constructs encoding drug resistant p38 (T106M) or BRAF (T529N) kinases. Parental HEPG2 cells used as control comparison. Cells were treated for 3 days. N = 3 independent experiments [mean, SEM]. c, Total and pT367 EZH2 levels in HEPG2 cells transduced with constructs encoding wildtype or drug resistant p38 (T106M) or BRAF (T529N) kinases. Parental HEPG2 cells used as control comparison. Cells were treated with DMSO, sorafenib (5 μM), or regorafenib (10 μM) for 1 hour. Endogenous antibodies used to assess overexpression of mutant constructs. d, Total and pT367 EZH2 levels in HEPG2 cells treated with DMSO, sorafenib, vemurafenib, or WNTinib at increasing doses (.01 μM to 30 μM) for 1 hour. Western blot results were independently validated at least two times. Extended data associated with Fig. 7.

Extended Data Fig. 9 |. WNTinib outperforms clinical compounds across in vivo models of HCC.

a-b, Dose escalation of WNTinib in BALB/c mice (a) and BALB/c/nude mice (b). Animals were dosed every day via oral gavage for 2 weeks. Percentage change in body weight noted in brackets (N = 3 animals per group; mean, SEM). c. Histological images (H&E) of liver, small & large intestine, and kidney from BALB/c mice treated with WNTinib at either 60 mg/kg or 120 mg/kg via oral gavage for 14 days. d, Images of C57BL/6 J mice treated with either vehicle or WNTinib for extended periods of time. WNTinib-treated animals present mosaic patterns of grey hair. e, Quantitative PCR expression of apoptotic genes in tumors derived from mice in panel Fig. 7e (N = 3 per group). *** P < .0005, as calculated with two-tailed paired t-tests. Exact P values listed in source data. For panel c, representative images are shown (n = 3). Extended data associated with Fig. 8.

Fig. 1 |. Chemical genetic screens in tumor organoids yield WNTinib as a selective antagonist of CTNNB1-mutated HCC.

a, Top, a probe set was derived from sorafenib and regorafenib, with points of diversification highlighted by R, which are specified for each analog in c. Bottom, viability of murine HCC organoid models (below: brightfield and histology) treated with the probe set or HCC-approved compounds. The 5-μM activity shown with endpoint viability was measured after 3 d (n = 2 independent experiments). Sorafenib, regorafenib, lenvatinib and cabozantinib were denoted by S, R, L and C, respectively. WNTinib and 8–50-2 are highlighted in red and turquoise, respectively. b, Chemical structures of WNTinib and 8–50-2. c, Left, IC₅₀ values for probe set and HCC-approved compounds in MYC-CTNNB1 tumor organoids, MYC-Tp53 tumor organoids and mpPHHs (n = 2 independent experiments). Right, WNT reporter expression levels in MYC-CTNNB1 tumor organoids treated with the same compounds (n = 3 independent experiments (mean, s.e.m.)). The R column illustrates the cap group diversity of analogs. WNTinib and 8–50-2 are highlighted in red and turquoise, respectively. d–f, IC₅₀ values for sorafenib, 8–50-2 or WNTinib in human HCC cell lines (d), primary human HCC cell lines (e) and primary human HCC organoids (f). CTNNB1 or WNT-pathway mutations are noted in the rightmost column (n = 3 independent experiments).

Fig. 2 |. WNTinib reduces EZH2 phosphorylation to drive the suppression of essential gene networks in CTNNB1-mutated HCC.

a, Volcano plots depicting phosphoproteomic changes elicited by WNTinib in MYC-CTNNB1 tumor organoids (left) or MYC-Tp53 tumor organoids (right) compared with DMSO. Inset, pathway enrichment terms associated with significantly regulated phosphoproteins. WNTinib was dosed at 1 μM for 24 h (n = 2 independent experiments). Gene-centered, differentially expressed phospho-sites were determined by using estimates of variance-mean dependence with a Benjamini–Hochberg FDR correction. FC, fold-change. b, Volcano plots depicting messenger RNA abundance changes elicited by WNTinib in MYC-CTNNB1 tumor organoids (left) or MYC-Tp53 tumor organoids (right) compared with DMSO. Inset, pathway enrichment terms associated with significantly regulated transcripts. WNTinib was dosed at 1 μM for 7 d (n = 2 independent experiments). Differential events were identified using two-sided Wald’s tests and Benjamini–Hochberg multiple-testing corrections. GO, gene ontology. c, Heatmaps and average profile plots for MYC-CTNNB1 tumor organoids (right) or MYC-Tp53 tumor organoids (left) displaying CUT&RUN H3K27me3 signal around the transcription start or end site (TSS/TES) (±3 kb) of genes downregulated by WNTinib in the MYC-CTNNB1 tumor organoids (RNA-seq, 7 d, b; n = 3,767 genes). d, As in c, but displaying H3K27me3 levels at genes belonging to the Wnt signaling pathway in WNTinib-treated MYC-CTNNB1 tumor organoids.

Fig. 3 |. WNTinib depends on EZH2 relocalization to chromatin for its activity in CTNNB1-mutated HCC.

a–c, Total and pT367 EZH2 levels in human liver cancer cell lines (a; matched to Fig. 1d), primary cell lines (b; matched to Fig. 1e) or organoids (c; matched to Fig. 1f) either mutated or WT for CTNNB1. Models were treated with sorafenib (5 μM), 8–50-2 (1 μM) or WNTinib (1 μM) for 24 h. d, Cytoplasmic and nuclear fractions of total and pT367 EZH2 from human HCC primary cell-line 23129 treated with DMSO, sorafenib (5 μM) or WNTinib (1 μM) for 24 h. Tubulin and histone H3 used as fractionation controls. e, Immunofluorescence images of EZH2 (green) localization in the human HCC primary cell-line 23129 treated with DMSO, sorafenib (5 μM), 8–50-2 (1 μM) or WNTinib (1 μM) for 24 h. DAPI (blue) was used as a nuclear stain and tubulin (TUBA, red) as a cytoplasmic stain. Scale bar, 40 μm. f, IC₅₀ curves for WNTinib (top) or sorafenib (bottom) in MYC-CTNNB1 tumor organoids depleted for EZH2 using CRISPR. Organoids were treated for 3 d. Inset, western blot depicting depletion efficiency (n = 3 independent experiments (mean, s.e.m.)). g,h, Combination treatment matrices for MYC-CTNNB1 tumor organoids treated with WNTinib (g) or sorafenib (h) and compounds targeting EZH2 (top, MS1943 EZH2 degrader; bottom, GSK343 EZH2 SAM competitive inhibitor). Tumor organoids were first treated with MS1943 or GSK343 for 3 d, followed by co-treatment with sorafenib or WNTinib for an additional 3 d (n = 2 independent experiments). CI values were calculated for each column and averaged across each matrix. Western blotting results were independently validated at least twice. For e, representative images are shown from n = 10 independent images captured from n = 2 independent experiments.

Fig. 4 |. WNTinib utilizes unique polypharmacology to regulate the EZH2–WNT axis.

a, Live-cell target engagement IC₅₀ values for sorafenib, regorafenib, 8–50-2 and WNTinib on clinically relevant receptor tyrosine kinases, cytoplasmic kinases and nonkinases. Above, RNA expression of targets in MYC-CTNNB1 and MYC-Tp53 tumor organoids. Heatmap displays averages from n = 3 independent experiments. FPKM, Fragment per kilobase of transcript per million mapped reads. b, Model. Left, phosphorylation of EZH2 is regulated downstream of an RTK KIT signaling axis with negative feedback mediated through cytoplasmic kinases, including BRAF and p38. Middle, sorafenib inhibition on upstream targets is mitigated due to release of negative feedback signaling through direct binding on BRAF and p38. Right, WNTinib strongly downregulates phosphor-EZH2 due to inhibition of targets and removal of anti-target inhibition, thereby avoiding compensatory feedback. As a result, unphosphorylated EZH2 localizes to the nucleus to repress the transcription of WNT targets.

Fig. 5 |. WNTinib utilizes unique polypharmacology to regulate the EZH2–WNT axis.

a, Time course (1–48 h) of signaling perturbations on relevant targets to the model shown in Fig. 4b (p-KIT, pMEK, p-p38, p-ATF2 and pT367 EZH2). MYC-CTNNB1 tumor organoids were treated with DMSO, sorafenib (10 μM), regorafenib (10 μM), 8–50-2 (1 μM), WNTinib (1 μM), PH-797804 (10 μM), vemurafenib (10 μM) or axitinib (5 μM). Western blotting measures endogenous proteins as indicated. b, In vitro kinase assay using either activated ERK or p38 kinases and an EZH2 peptide encoding the T367 site. c, In vitro kinase assay conducted as in b, but using a set concentration of the EZH2 peptide (1 μM). Relevant inhibitors (SHC772984, PH-797804 or WNTinib; all used at 1 μM) were used to gauge target engagement and concomitant suppression of pT367 EZH2. d, Total and pT367 EZH2 levels in HEPG2 cells transduced with constructs encoding constitutively active MEK (Ser218Asp, Ser222Asp) or MKK6 (Ser207Glu, Thr211Glu). Parental HEPG2 cells were used as a control comparison. Cells were treated with DMSO, sorafenib (5 μM), regorafenib (10 μM), 8–50-2 (0.5 μM), WNTinib (0.5 μM), PH-797804 (10 μM), vemurafenib (10 μM) or axitinib (5 μM) for 24 h. The V5 antibody was used to assess overexpression of mutant constructs. e, IC₅₀ curves for WNTinib (top) or sorafenib (bottom) in HEPG2 cells transduced with constructs encoding constitutively active MEK (Ser218Asp, Ser222Asp) or MKK6 (Ser207Glu, Thr211Glu). Parental HEPG2 cells were used as a control comparison. Cells were treated for 3 d (n = 3 independent experiments (mean, s.e.m.)). Western blotting results were independently validated at least twice.

Fig. 6 |. KIT is a critical target for WNTinib’s MoA.

a, Model of KIT bound to WNTinib. The inactive type II conformation enables binding through the p-C₂F₅ group, which is highlighted with a red sphere. b, Growth curves for MYC-CTNNB1 tumor organoids (left) or MYC-Tp53 tumor organoids (right) treated with increasing doses of cKIT ligand/SCF (n = 3 independent experiments (mean, s.e.m.)). Significant differences between curves (compared with untreated) are indicated by asterisks: ***P < 0.0005, as calculated using a two-way ANOVA and Tukey’s test for multiple comparisons (F[4, 20] = 7.1). c, Combination treatment matrices for MYC-CTNNB1 tumor organoids treated with WNTinib (left) or sorafenib (right) and SCF. Tumor organoids were treated for 3 d (n = 2 independent experiments). CI values were calculated for each column and averaged across each matrix. d, Western blot depicting the modulation of pT367 EZH2 by increasing titration of SCF in MYC-CTNNB1 tumor organoids treated with sorafenib (10 μM), 8–50-2 (1 μM) or WNTinib (1 μM) for 24 h. e, Total and pT367 EZH2 levels in HEPG2 cells transduced with a doxycycline (dox)-inducible construct encoding constitutively active cKIT (Val559Asp, Thr670Ile). Cells were treated with DMSO, sorafenib (5 μM), regorafenib (10 μM), 8–50-2 (0.5 μM), WNTinib (0.5 μM), PH-797804 (10 μM), vemurafenib (10 μM) or axitinib (5 μM) for 24 h. f, IC₅₀ curve for WNTinib in HEPG2 cells transduced as in e. Doxycycline-untreated HEPG2 cells were used as a control comparison. Cells were treated for 3 d (n = 3 independent experiments (mean, s.e.m.)). g, WNT reporter expression levels in HEPG2 cells transduced with the cKIT-V559D, T670I construct and treated with WNTinib (0.5 μM) for 24 h (n = 3 independent experiments (mean, s.e.m.)). Significant differences between groups are indicated by asterisks.: *P < 0.05, **P < 0.005 as calculated using two-tailed, paired Student’s t-tests. For b, left: P values: ***P < 0.0001 (10, 30 and 100 ng μl⁻ of SCF). For g, P values: *P = 0.009 (−dox, +WNTinib), **P = 0.0012 (+dox, +WNTinib). Western blotting results were independently validated at least twice.

Fig. 7 |. BRAF and p38 kinases are critical anti-targets for WNTinib.

a, Total and pT367 EZH2 levels in HEPG2 cells transduced with constructs encoding drug-resistant p38 (Thr106Met) or BRAF (Thr529Asn) kinases. Parental HEPG2 cells were used as control comparison. Cells were treated with DMSO, sorafenib (5 μM), regorafenib (10 μM), 8–50-2 (0.5 μM), WNTinib (0.5 μM), PH-797804 (10 μM), vemurafenib (10 μM) or axitinib (5 μM) for 24 h. V5 and endogenous antibodies were used to assess overexpression of mutant constructs. b, IC₅₀ curve for WNTinib in HEPG2 cells transduced with constructs encoding drug-resistant p38 (Thr106Met) or BRAF (Thr529Asn) kinases. Parental HEPG2 cells were used as a control comparison. Cells were treated for 3 d (n = 3 independent experiments (mean, s.e.m.)). c,d, Combination treatment matrices for MYC-CTNNB1 tumor organoids treated with WNTinib (left) or sorafenib (right) and the BRAF inhibitor dabrafenib (c) or the p38 inhibitor SB202190 (d). Tumor organoids were treated for 3 d (n = 2 independent experiments). CI values were calculated for each column and averaged across each matrix. e, Western blot depicting the modulation of pT367 EZH2 by WNTinib (1 μM) alone or in combination with dabrafenib (10 μM), SB202190 (10 μM) or the two compounds together. MYC-CTNNB1 tumor organoids were treated for 24 h. f, WNT reporter expression levels in MYC-CTNNB1 tumor organoids treated with WNTinib alone (1 μM) or in combination with dabrafenib (10 μM) or SB202190 (10 μM) for 24 h (n = 3 independent experiments (mean, s.e.m.)). Significant differences between groups are indicated by asterisks: *P < 0.05, ^**P < 0.005, ***P < 0.0005, as calculated using two-tailed, paired Student’s t-tests: ***P = 0.00039 (WNTinib), **P = 0.0036 (WNTinib + BRAFi), *P = 0.0168 (WNTinib + P38i). Western blot results were independently validated at least twice.

Fig. 8 |. WNTinib outperforms clinical compounds across in vivo models of HCC.

a, Dose escalation of WNTinib in C57BL/6J mice. The percentage change in body weight was noted in brackets (n = 3 animals per group (mean, s.e.m.)). b, Pharmacokinetic curves of WNTinib, sorafenib and regorafenib in BALB/c animals. For reference, WNTinib’s in vitro IC₅₀ (1 μM) was denoted with a dashed line. c, Left, tumor volume plot in MYC-CTNNB1 tumor organoid allografts treated with WNTinib or clinical compounds (n = 10 animals per group for WNTinib and vehicle; n = 5 animals per group for clinicals (mean, s.e.m.)). Right, waterfall plot. Significant differences between groups are indicated by asterisks: *P < 0.05, as calculated using a two-way ANOVA with Tukey’s test for multiple comparisons (F[5,36] = 3.349). d, As in c, but using MYC-Tp53 tumor organoid allografts (n = 5 animals per group (mean, s.e.m.)). Significant differences between groups are indicated by asterisks: *P < 0.05, as calculated using a two-way ANOVA with Tukey’s test for multiple comparisons (F[5, 36] = 3.485). For c and d, the animals were dosed via daily oral gavage using 30 mg kg⁻¹ of the respective compounds. e, Tumor volume plot in MYC-CTNNB1 tumor organoid allografts treated with WNTinib or sorafenib (n = 9 vehicle; n = 10 sorafenib; n = 11 WNTinib (mean, s.e.m.)). Significant differences between groups are indicated by asterisks: **P < 0.005, ***P < 0.0005, as calculated using a two-way ANOVA with Tukey’s test for multiple comparisons (F[4,216] = 4.272). f, The qPCR expression of WNT-target genes in tumors derived from mice in e (n = 3 per group). *P < 0.05, as calculated using two-tailed, paired Student’s t-tests. g, Western blot of pT367 EZH2 in tumors derived from mice in e (n = 3 per group). h, Hydrodynamic tail-vein model of CTNNB1-mutated HCC (MYC-lucOS; CTNNB1) treated with vehicle, sorafenib or WNTinib. Percentage survival is shown and log(rank P) values are indicated (compared with vehicle). Kinase inhibitors were started 7 d post-injection and dosed at 20 mg kg⁻¹ (WNTinib) or 30 mg kg⁻¹ (sorafenib)—5 d on and 2 d off. Exact P values are listed in Source data.