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.
