Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.can-23-2081

Figure Lengend Snippet: Figure 1. Epi-Drug CRISPR dropout screens identify RUVBL1/2 as vulnerabilities of YTHDF1-expressing colorectal cancer cells. A, Composition of Epi-Drug sgRNA library and the workflow of CRISPR-Cas9 screens to identify YTHDF1-dependent vulnerabilities in colorectal cancer cells. B, Principal component analysis (PCA) of sgRNA abundances in each group at the end point of CRISPR-Cas9 screening. C, Left, top depleted genes in YTHDF1-overexpressing DLD1 cells vs. control vector (log2(fold change) < 0.5; log10(P value < 1). Middle, top enriched genes in shYTHDF1 cells vs. shControl (log2(fold change) > 0.5; log10(P value < 1). Right, overlapping of outlier genes identified the common candidates preferentially essential in a YTHDF1-dependent fashion. D and E, RUVBL1/2 mRNA expression in colorectal cancer cells compared with adjacent normal tissues in Hong Kong (D) and TCGA (E) colorectal cancer cohorts. In Hong Kong cohort, mRNA expression was normalized to β-actin. F, RUVBL1/2 and YTHDF1 proteins are overexpressed in colorectal cancer cells compared with paired adjacent normal tissues. G, Left, representative images of YTHDF1, RUVBL1, and RUVBL2 staining in colorectal cancer tissue microarrays (N ¼ 184). Right, Pearson correlation analysis of YTHDF1, RUVBL1, and RUVBL2 protein expression. H, Left, Kaplan–Meier curve analysis of RUVBL1 protein expression and patient survival in colorectal cancer in tissue microarray cohort (N ¼ 184). Right, multivariate Cox regression analysis. RUVBL1-low, IHC score 1; RUVBL1-high, IHC score 2 to 3. I, Left, Kaplan–Meier curve analysis of RUVBL2 protein expression and colorectal cancer patient survival. Right, multivariate Cox regression analysis. RUVBL2-low, IHC score 1 to 2; RUVBL2-high, IHC score 3. Paired t test (D and E; left), Student t-test (E; right), Pearson χ2 test (G), or log rank test (H and I).

Article Snippet: For recombinant protein pulldown, human YTHDF1 (Origene, TP307185L), RUVBL1 (Origene, TP301170), RUVBL2 (Origene, TP300933) proteins (2 μg) were incubated in stock buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, pH 8.0) for overnight at 4°C, followed by pulldown assays as described above.

Techniques: CRISPR, Expressing, Control, Plasmid Preparation, Staining, Microarray

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.can-23-2081

Figure Lengend Snippet: Figure 4. YTHDF1 promotes translation efficiency of RUVBL1/2, which in turn interact with YTHDF1 and translational initiation factors. A, RNC-qPCR analysis of ribosome- associated RUVBL1/2 mRNA in vector- and YTHDF1-overexpressing DLD1 and HCT116 cells. B, Enrichment of RUVBL1/2 mRNA in < 40S, 40S, 60S, 80S, and polysomes from HCT116 cells with or without YTHDF1 overexpression. C and D, Colorectal cancer cells overexpressing wildtype YTHDF1 or mutant YTHDF1 were transfected with pmirGLO-RUVBL1 (C) or pmirGLO-RUVBL2 (D) containing respective 30UTR sequences, followed by luciferase assays. E, pmirGLO-RUVBL1/2-mutant reporters with mutated m6A sites (RRACH to TTTCT) in the 30UTR region demonstrated decreased luciferase activity. F, RUVBL1/2 coimmunoprecipitation and mass spectrometry for identification of common interacting proteins. G, Pathway enrichment analysis [gene ontology (GO), GSEA-KEGG] of interacting partners of RUVBL1/2. H, Coimmunoprecipitation by anti-YTHDF1 verified binding of YTHDF1 to RUVBL1/2. I, Coimmunoprecipitation using recombinant YTHDF1 and RUVBL1/2 confirmed direct protein–protein interplay between YTHDF1 and RUVBL1/2. J, Colocalization of RUVBL1/2 and YTHDF1 in HCT116 cells was determined by immu- nofluorescence staining. Student t test (A, B, and E) and one-way ANOVA (C and D). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: For recombinant protein pulldown, human YTHDF1 (Origene, TP307185L), RUVBL1 (Origene, TP301170), RUVBL2 (Origene, TP300933) proteins (2 μg) were incubated in stock buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, pH 8.0) for overnight at 4°C, followed by pulldown assays as described above.

Techniques: Plasmid Preparation, Over Expression, Mutagenesis, Transfection, Luciferase, Activity Assay, Mass Spectrometry, Binding Assay, Recombinant, Staining

Journal: Cancer Research

Article Title: RUVBL1/2 Blockade Targets YTHDF1 Activity to Suppress m6A-Dependent Oncogenic Translation and Colorectal Tumorigenesis

doi: 10.1158/0008-5472.can-23-2081

Figure Lengend Snippet: Figure 6. Pharmacological RUVBL1/2 inhibitor inhibits the growth of YTHDF1-overexpressing colorectal cancer cells. A, Structure of a RUVBL1/2 complex inhibitor, CB6644. B, Forty-eight hours-IC50 values indicated that CB6644 preferentially inhibited the growth of DLD1 and HCT116 cells with YTHDF1 overexpression. C, CB6644 preferentially impaired colony formation capacity in YTHDF1-overexpressing DLD1 and HCT116 cells (7–14 days). D, CB6644 (0.5 µmol/L for DLD1; 0.1 µmol/L for HCT116, 24 hours) abrogated suppressive effect of YTDHF1 overexpression on apoptosis. Puromycin (0.5 µg/mL, 24 hours) was used as positive control. E, Treatment of DLD1 cells with CB6644 (0.5 µmol/L, 36 hours), followed by coimmunoprecipitation to analyze their interactions with YTHDF1. F, Interaction between YTHDF1 and EIF3K or EIF4A after treatment with CB6644 in DLD1 cells (0.5 µmol/L, 36 hours). G, Effect of CB6644 on protein translation in DLD1 cells, as assessed by puromycin incorporation assay (0.5 µmol/L, 6 hours). H, DLD1 cells expressing sgRUVBL1 or sgRUVBL2 were overexpressed with wildtype or ATPase-dead mutant RUVBL1 or RUVBL2, respectively. Coimmunoprecipitation was performed with anti-YTHDF1 to determine its interaction with RUVBL1/2, EIF3K, and EIF4A. I, Effect of ATPase-dead mutant RUVBL1 or RUVBL2 on protein translation in DLD1 cells compared with wildtype counterparts. One-way ANOVA (E and F). ****, P < 0.0001.

Article Snippet: For recombinant protein pulldown, human YTHDF1 (Origene, TP307185L), RUVBL1 (Origene, TP301170), RUVBL2 (Origene, TP300933) proteins (2 μg) were incubated in stock buffer (50 mmol/L Tris-Cl, 150 mmol/L NaCl, pH 8.0) for overnight at 4°C, followed by pulldown assays as described above.

Techniques: Over Expression, Positive Control, Expressing, Mutagenesis