FGFR4 inhibitor treatment elevates EZH2 expression by activating non-canonical NF-kB signaling in HCC. A Western bolt detected the expression of EZH2 and FGFR1-4 in human hepatic cell line THLE-2 and human hepatoma cell lines HepG2, SMMC-7721 and MHCC97L. B Dose responses of zebrafish KRAS^G12V+ HCC primary tumors treated with Roblitinib for 72 h. Scale bars: 100 ?m. Data are presented as mean ± SEM (n = 15, one-way ANOVA with Dunnett?s multiple comparison test, **p < 0.01, ns, no significance). C Clustered heatmaps showed the differentially expressed genes between control group and Roblitinib treatment group (fold change ? 2 or ? ? 2 and p-values < 0.05). HepG2 cells were treated with Roblitinib for 48 h, followed by RNA-Seq analysis. Red indicates high relative expression, and blue indicates low relative expression. D Bubble chart showed the Gene Ontology (GO) biological process and molecular function analysis of differentially expressed genes following Roblitinib treatment in (C). P-values < 0.05 was regarded as statistically significant. E Gene set enrichment analysis (GSEA) of the KEGG pathway showed that HCC cells treated with Roblitinib were enriched for the drug resistance/antagonism. F GSEA of Reactome pathway showed that cells treated with Roblitinib were enriched for transcriptional signatures associated with PRC2-mediated methylation. G-H Western bolt detected the expression of EZH2 after different FGFR inhibitor treatment for 48 h (G) and Roblitinib treatment for different durations (H). I High levels of EZH2 were associated with worse survival of HCC patients. J Gene set enrichment analysis (GSEA) showed that the NF-kB signaling pathway was enriched in the Roblitinib treatment group compared with Control group. K RNA-Seq analysis of the expression level of the indicated NF-kB genes after Roblitinib treatment for 48 h. The abscissa represented the indicated NF-kB genes, while the ordinate represented Fragments Per Kilobase of exon model per Million mapped fragments (FPKM). Data are presented as mean ± SEM (n = 3, two-way ANOVA with Sidak?s multiple comparison test, ****p < 0.0001, ns, no significance). L Western bolt detected the expression of the indicated NF-kB genes after Roblitinib treatment for 48 h. M Western blot analysis of EZH2 expression in HepG2 cells transfected with empty vector control (Con), HA-tagged NFKB2 (HA-NFKB2) and NFKB2-shRNAs (shNFKB2-1 and 2). N Western blot analysis of EZH2 expression in HepG2 cells after lentiviral transduction with control shRNA (shVector) or 3? UTR NFKB2-targeting shRNA (shNFKB2) and restored with empty vector control (Con) or HA-tagged NFKB2 (HA-NFKB2), following by Roblitinib treatment for 48 h

Elevated EZH2 levels lead to antagonism of HCC against FGFR4 inhibitors. A Western blot analysis of EZH2 expression in HepG2 cells transfected with empty vector control (Con), FLAG-EZH2 (EZH2-Ov) and EZH2-siRNAs (EZH2-siRNA1 and 2). B Cell viability of HepG2 cells transfected with empty vector control (Con), FLAG-EZH2 (EZH2-Ov) and EZH2-siRNAs (EZH2-siRNA1 and 2) was evaluated by the CCK-8 following increasing concentrations of Roblitinib treatment for 48 h. Data are presented as mean ± SEM (n = 3). C Crystal violet staining of HepG2 and Huh7 cell lines transfected with empty vector control (Con), FLAG-EZH2 (EZH2-Ov) and EZH2-siRNAs (EZH2-Kd) following Roblitinib treatment for 48 h. Scale bars: 1 cm. D EdU assays of HepG2 cells transfected with empty vector control (Con), FLAG-EZH2 (EZH2-Ov) and EZH2-siRNAs (EZH2-Kd) following Roblitinib treatment for 48 h. Scale bars: 100 ?m. E Measurement of the cell numbers in (D). Data are presented as mean ± SEM (n = 3, two-way ANOVA with Sidak?s multiple comparison test, ***p < 0.001, ****p < 0.0001, ns, no significance). F Dose responses of zebrafish KRAS^G12V+/EZH2⁺ (top) and KRAS^G12V+ (bottom) HCC primary tumors treated with Roblitinib for 72 h. Scale bars: 100 ?m. G Measurement of the tumor sizes in (F). Data are presented as mean ± SEM (n = 15, two-way ANOVA with Sidak?s multiple comparison test, *p < 0.05, ns, no significance)

Combination of Roblitinib and CPI-169 synergistically inhibits the HCC cell growth. A-C Cell viability of HepG2 (A), SMMC-7721 (B) and MHCC97H (C) cell lines was evaluated by the CCK-8 following increasing concentrations of CPI-169, Roblitinib or CPI-169 + Roblitinib treatment for 48 h. Data are presented as mean ± SEM (n = 3). D Drug interaction analysis between CPI-169 and Roblitinib in HepG2, SMMC-7721, MHCC97H, MHCC97L and Huh7 cell lines. The CI values less than 1.0, approximately 1.0, and greater than 1.0 indicate synergism, additive, and antagonism, respectively. E EdU assay of HepG2 cells following CPI-169, Roblitinib or CPI-169 + Roblitinib treatment for 48 h. Scale bars: 50 ?m. F Colony formation assay of HepG2 and SMMC-7721 cell lines following CPI-169, Roblitinib or CPI-169 + Roblitinib treatment for 14 days. Scale bars: 1 cm. G Measurement of the cell numbers in (E). Data are presented as mean ± SEM (n = 3, one-way ANOVA with Tukey?s multiple comparison test, *p < 0.05, ***p < 0.001, ****p < 0.0001, ns, no significance). H Measurement of the clone numbers in (F). Data are presented as mean ± SEM (n = 3, two-way ANOVA with Sidak?s multiple comparison test, ****p < 0.0001, ns, no significance). I Western blot analysis of the indicated protein expression in HepG2 cells following CPI-169, Roblitinib or CPI-169 + Roblitinib treatment for 48 h. J-K Zebrafish harboring KRAS^G12V+ HCC primary tumors (J) and zebrafish HCC xenografts using the mCherry-labbled HepG2 cells (K) treated with CPI-169, Roblitinib or CPI-169 + Roblitinib for 72 h. Scale bars: 100 ?m. Data are presented as mean ± SEM (n = 15, one-way ANOVA with Tukey?s multiple comparison test, *p < 0.05, ***p < 0.001, ****p < 0.0001, ns, no significance)

Combination of Roblitinib and CPI-169 elicits robust anti-cancer effects in vivo. A-C Subcutaneous xenograft tumors established from SMMC-7721 cells were treated with vehicle or the indicated drugs. Shown are the tumors (A), tumor weight (B) and the tumor volume (C) from the recipient mice. Mice with SMMC-7721 xenografts were treated with vehicle (n = 6), CPI-169 (n = 6), Roblitinib (n = 6) or CPI-169 + Roblitinib (n = 6) for 2 weeks. Scale bars: 1 cm. Values are presented as mean ± SEM; One-way ANOVA with Tukey?s multiple comparisons test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. D Representative H&E and IHC staining for H3K27me3, phospho-FGFR4 (p-FGFR4), Ki67 and Cleaved caspase-3 (cl. Caspase 3) in tumors. Scale bars: 50 ?m. The intensity of Ki67 and Cleaved caspase-3 staining cells were quantified. Values are presented mean ± SEM; One-way ANOVA with Tukey?s multiple comparisons test, ***p < 0.001, ****p < 0.0001

Combination of Roblitinib and CPI-169 synergistically inhibits the YAP signaling. A Volcano Plot showed the differentially expressed genes between control group and CPI-169 + Roblitinib treatment group (fold change ? 2 or ? ? 2 and p-values < 0.05). HepG2 cells were treated with CPI-169 + Roblitinib for 48 h, followed by RNA-Seq analysis. Red indicates high relative expression, and blue indicates low relative expression. B Bubble chart showed the KEGG pathway analysis of differentially expressed genes following CPI-169 + Roblitinib treatment in (A). P-values < 0.05 was regarded as statistically significant. C Gene set enrichment analysis (GSEA) showed that the Hippo signaling pathway was enriched in the co-treatment group compared with Control group. D Western bolt detected the expression level of YAP1 and p-YAP1 after CPI-169, Roblitinib or CPI-169 + Roblitinib treatment for 48 h. E Venn diagram showed YAP1 target genes that were regulated by CPI-169 + Roblitinib treatment. F Gene functional annotation of GO enrichment analysis of the 141 down-regulated YAP1 target genes in (E) via Metascape. G Heatmap representing the expression levels of 18 YAP1 target genes related to cell proliferation, cell migration and anti-apoptosis determined by RNA-Seq in HepG2 cells following CPI-169, Roblitinib or CPI-169 + Roblitinib treatment for 48 h. H Genomic tracks displayed ChIP-seq data for YAP1 around the indicated genes and their corresponding RNA-Seq signals in (G). I qPCR analysis of the representative YAP1 target genes from typical pathway related to cell proliferation, cell migration and anti-apoptosis in (G). Data are presented as mean ± SEM (n = 3, one-way ANOVA with Tukey?s multiple comparisons test, *P < 0.05, **P < 0.01, ***p < 0.001, ****p < 0.0001, ns, no significance). J Luciferase assay for YAP/TAZ activity in HepG2 and SMMC-7721 cells following CPI-169, Roblitinib or CPI-169 + Roblitinib treatment for 48 h. Data are presented as mean ± SEM (n = 3, two-way ANOVA with Sidak?s multiple comparison test, **P < 0.01, ****p < 0.0001)

Overexpression of YAP1^S127A antagonizes the synergistic inhibitory effect of Roblitinib and CPI-169 in HCC cells. A Western blot analysis of FLAG-YAP1^S127A expression in HepG2 and SMMC-7721 cell lines stably transfected with empty vector control (Con) or FLAG-YAP1^S127A (YAP1^S127A). B qPCR analysis of YAP1 mRNA level in HepG2 and HepG2 YAP1^S127A cell lines following the CPI-169 + Roblitinib treatment for 48 h. Data are presented as mean ± SEM (n = 3, two-way ANOVA with Sidak?s multiple comparison test, ****p < 0.0001, ns, no significance). C Cell viability of HepG2, HepG2 YAP1^S127A, SMMC-7721 and SMMC-7721 YAP1^S127A cell lines was evaluated by the CCK-8 following increasing concentrations of CPI-169 + Roblitinib treatment for 48 h. Data are presented as mean ± SEM (n = 3). D Drug interaction analysis between CPI-169 and Roblitinib in HepG2, HepG2 YAP1^S127A, SMMC-7721 and SMMC-7721 YAP1^S127A cell lines. The CI values less than 1.0, approximately 1.0 and greater than 1.0 indicate synergism, additive and antagonism, respectively. E EdU assays of HepG2 and HepG2 YAP1^S127A cell lines following the CPI-169 + Roblitinib treatment for 48 h. Scale bars: 50 ?m. F Colony formation assay of HepG2 and HepG2 YAP1^S127A cell lines following CPI-169, Roblitinib or CPI-169 + Roblitinib treatment for 14 days. Scale bars: 1 cm. G Measurement of the cell numbers in (E). Data are presented as mean ± SEM (n = 3, two-way ANOVA with Sidak?s multiple comparison test, ***p < 0.001, ****p < 0.0001). H Measurement of the clone numbers in (F) and SMMC-7721 and SMMC-7721 YAP1^S127A cell lines following CPI-169 and/or Roblitinib treatment for 14 days. Data are presented as mean ± SEM (n = 3, two-way ANOVA with Sidak?s multiple comparison test, *p < 0.05, ***p < 0.001, ****p < 0.0001, ns, no significance). I-J Flow cytometric analysis of apoptosis in HepG2 and HepG2 YAP1^S127A cell lines following CPI-169 + Roblitinib treatment for 48 h. Data are presented as mean ± SEM (n = 3, two-way ANOVA with Sidak?s multiple comparison test, **p < 0.01, ****p < 0.0001). K-L Zebrafish harboring KRAS^G12V+ (left) or KRAS^G12V+/YAP1^S87A+ (right) HCC primary tumors were treated with CPI-169 + Roblitinib for 72 h. Scale bars: 100 ?m. Data are presented as mean ± SEM (n = 12, two-way ANOVA with Sidak?s multiple comparison test, *p < 0.05, ****p < 0.0001 ns, no significance)

Proposed model for synergetic induction of apoptosis in HCC by FGFR4 and EZH2 inhibitors. Treatment with FGFR4 inhibitors alone induced EZH2 accumulation by activating NF-kB2, which maintained YAP expression levels to maintain some degree of cell proliferation-, migration- and anti-apoptosis-related genes expression, resulting in antagonism to Roblitinib. Only when FGFR4 and EZH2 were simultaneously suppressed would YAP expression and its corresponding signaling decrease sharply and eventually synergistically induce substantial HCC cell apoptosis